Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids

ABSTRACT

The present invention is directed to an inventive polymeric carrier molecule according to generic formula (I) and variations thereof, which allows for efficient transfection of nucleic acids into cells in vivo and in vitro, a polymeric carrier cargo complex formed by a nucleic acid and the inventive polymeric carrier molecule, but also to methods of preparation of this inventive polymeric carrier molecule and of the inventive polymeric carrier cargo complex. The present invention also provides methods of application and use of this inventive polymeric carrier molecule and the inventive polymeric carrier cargo complex as a medicament, for the treatment of various diseases, and in the preparation of a pharmaceutical composition for the treatment of such diseases.

The present invention is directed to an inventive polymeric carriermolecule according to generic formula (I) and variations thereof, whichallows for efficient transfection of nucleic acids into cells in vivoand in vitro, a polymeric carrier cargo complex formed by a nucleic acidand the inventive polymeric carrier molecule, but also to methods ofpreparation of this inventive polymeric carrier molecule and of theinventive polymeric carrier cargo complex. The present invention alsoprovides methods of application and use of this inventive polymericcarrier molecule and the inventive polymeric carrier cargo complex as amedicament, for the treatment of various diseases, and in thepreparation of a pharmaceutical composition for the treatment of suchdiseases.

Various diseases today require a treatment which involves administrationof peptide-, protein-, and nucleic acid-based drugs, particularly thetransfection of nucleic acids into cells or tissues. The fulltherapeutic potential of peptide-, protein-, and nucleic acid-baseddrugs is frequently compromised by their limited ability to cross theplasma membrane of mammalian cells, resulting in poor cellular accessand inadequate therapeutic efficacy. Today this hurdle represents amajor challenge for the biomedical development and commercial success ofmany biopharmaceuticals (see e.g. Foerg and Merkle, Journal ofPharmaceutical Sciences, published online at www.interscience.wiley.com,2008, 97(1): 144-62).

For some diseases or disorders, gene therapeutic approaches have beendeveloped as a specific form of such treatments. These treatments ingeneral utilize transfection of nucleic acids or genes into cells ortissues, whereas gene therapeutic approaches additionally involve theinsertion of one or more of these nucleic acids or genes into anindividual's cells and tissues to treat a disease, e.g. hereditarydiseases, in which a defective mutant allele is replaced with afunctional one.

Transfer or insertion of one or more of these nucleic acids or genesinto an individual's cells, however, still represents a major challengetoday and is absolutely necessary for ensuring a good therapeuticaleffect of a nucleic acid based medicaments, particularly in the field ofgene therapy.

To achieve successful transfer of nucleic acids or genes into anindividual's cells, a number of different hurdles have to be passed. Thetransport of nucleic acids typically occurs via association of thenucleic acid with the cell membrane and subsequent uptake by theendosomes. In the endosomes, the introduced nucleic acids are separatedfrom the cytosol. As expression occurs in the cytosol, these nucleicacids have to depart the cytosol. If the nucleic acids do not managedeparting the cytosol, either the endosome fuses with the lysosomeleading to a degradation of its content, or the endosome fuses with thecell membrane leading to a return of its content into the extracellularmedium. For efficient transfer of nucleic acids, the endosomal escapethus appears to be one of the most important steps additional to theefficiency of transfection itself. Until now, there are differentapproaches addressing these issues. However, no approach was at leastsuccessful in all aspects.

Transfection agents used in the art today typically comprise peptides,different polymers, lipids as well as nano- and microparticles (see e.g.Gao, X., K. S. Kim, et al. (2007), Aaps J 9(1): E92-104). Thesetransfection agents typically have been used successfully only in invitro reactions. When transfecting nucleic acids in vivo into cells of aliving animal, further requirements have to be fulfilled. As an example,the complex has to be stable in physiological salt solutions withrespect to agglomerisation. Furthermore, it does not interact with partsof the complement system of the host. Additionally, the complex shallprotect the nucleic acid from early extracellular degradation byubiquitiously occurring nucleases. For genetherapeutic applications itis furthermore of utmost importance, that the carrier is not recognizedby the adaptive immune system (immunogenicity) and does not stimulate anunspecific cytokine storm (acute immune response) (see Gao, Kim et al.,(2007, supra); Martin, M. E. and K. G. Rice (2007), Aaps J 9(1): E18-29;and Foerg and Merkle, (2008, supra)). Foerg and Merkle (2008, supra),discuss therapeutic potential of peptide-, protein and nucleicacid-based drugs. According to their analysis, the full therapeuticpotential of these drugs is frequently compromised by their limitedability to cross the plasma membrane of mammalian cells, resulting inpoor cellular access and inadequate therapeutic efficacy. Today thishurdle represents a major challenge for the biomedical development andcommercial success of many biopharmaceuticals.

In this context, Gao et al. (Gao et al. The AAPS Journal 2007; 9(1)Article 9) see the primary challenge for gene therapy in the developmentof a method that delivers a therapeutic gene to selected cells whereproper gene expression can be achieved. Gene delivery and particularlysuccessful transfection of nucleic acids into cells or tissue is,however, not simple and typically dependent on many factors. Forsuccessful delivery, e.g., delivery of nucleic acids or genes into cellsor tissue, many barriers must be overcome. According to Gao et al.(2007) an ideal gene delivery method needs to meet 3 major criteria: (1)it should protect the transgene against degradation by nucleases inintercellular matrices, (2) it should bring the transgene across theplasma membrane and (3) it should have no detrimental effects.

These goals may be achieved by using a combination of differentcompounds or vectors. Notably, there are some compounds or vectors,which overcome at least some of these barriers.

Most usually, transfection, e.g. of nucleic acids, is carried out usingviral or non-viral vectors. For successful delivery, these viral ornon-viral vectors must be able to overcome the above mentioned barriers.The most successful gene therapy strategies available today rely on theuse of viral vectors, such as adenoviruses, adeno-associated viruses,retroviruses, and herpes viruses. Viral vectors are able to mediate genetransfer with high efficiency and the possibility of long-term geneexpression, and satisfy 2 out of 3 criteria. However, the acute immuneresponse, immunogenicity, and insertion mutagenesis uncovered in genetherapy clinical trials have raised serious safety concerns about somecommonly used viral vectors.

A solution to this problem may be found in the use of non-viral vectors.Although non-viral vectors are not as efficient as viral vectors, manynon-viral vectors have been developed to provide a safer alternative ingene therapy. Methods of nonviral gene delivery have been explored usingphysical (carrier-free gene delivery) and chemical approaches (syntheticvector-based gene delivery). Physical approaches usually include needleinjection, electroporation, gene gun, ultrasound, and hydrodynamicdelivery, employ a physical force that permeates the cell membrane andfacilitates intracellular gene transfer. The chemical approachestypically use synthetic or naturally occurring compounds (cationiclipids, cationic polymers, lipid-polymer hybrid systems) as carriers todeliver the transgene into cells. Although significant progress has beenmade in the basic science and applications of various nonviral genedelivery systems, the majority of nonviral approaches is still much lessefficient than viral vectors, especially for in vivo gene delivery (seee.g. Gao et al. The AAPS Journal 2007; 9(1) Article 9).

Over the past decade, attractive prospects for a substantial improvementin the cellular delivery of nucleic acids have been announced that weresupposed to result from their physical assembly or chemical ligation toso-called cell penetrating peptides (CPPs) also denoted asprotein-transduction domains (PTDs) (see Foerg and Merkle, (2008,supra)). CPPs represent short peptide sequences of 10 to about 30 aminoacids which can cross the plasma membrane of mammalian cells and maythus offer unprecedented opportunities for cellular drug delivery.Nearly all of these peptides comprise a series of cationic amino acidsin combination with a sequence, which forms an α-helix at low pH. As thepH is continuously lowered in vivo by proton pumps, a conformationalchange of the peptide is usually initiated rapidly. This helix motifmediates an insertion into the membrane of the endosome leading to arelease of its content into the cytoplasma (see Foerg and Merkle, (2008,supra); and Vives, E., P. Brodin, et al. (1997). “A truncated HIV-1 Tatprotein basic domain rapidly translocates through the plasma membraneand accumulates in the cell nucleus.” J Biol Chem 272(25): 16010-7).Despite these advantages, a major obstacle to CPP mediated drug deliveryis thought to consist in the often rapid metabolic clearance of thepeptides when in contact or passing the enzymatic barriers of epitheliaand endothelia. In conclusion, metabolic stability of CPPs represents animportant biopharmaceutical factor for their cellular bioavailability.However, there are no CPPs available in the art, which are on the onehand side stable enough to carry their cargo to the target before theyare metabolically cleaved, and which on the other hand side can becleared from the tissue before they can accumulate and reach toxiclevels.

One further approach in the art for delivering cargo molecules intocells, e.g. for gene therapy, comprises peptide ligands (see Martin andRice (see Martin and Rice, The AAPS Journal 2007; 9 (1) Article 3)).Peptide ligands can be short sequences taken from larger proteins thatrepresent the essential amino acids needed for receptor recognition,such as EGF peptide used to target cancer cells. Other peptide ligandshave been identified including the ligands used to target thelectin-like oxidized LDL receptor (LOX-1). Up-regulation of LOX-1 inendothelial cells is associated with dysfunctional states such ashypertension and atherosclerosis. Such peptide ligands, however, are notsuitable for many gene therapeutic approaches, as they cannot be linkedto their cargo molecules by complexation or adhesion but requirecovalent bonds, e.g. crosslinkers, which typically exhibit cytotoxiceffects in the cell.

Synthetic vectors may also be used in the art for delivering cargomolecules into cells, e.g., for the purpose of gene therapy. However,one main disadvantage of many synthetic vectors is their poortransfection efficiency compared to viral vectors and significantimprovements are required to enable further clinical development.Several barriers that limit nucleic acid transfer both in vitro and invivo have been identified, and include poor intracellular delivery,toxicity and instability of vectors in physiological conditions (see.e.g. Read, M. L., K. H. Bremner, et al. (2003). “Vectors based onreducible polycations facilitate intracellular release of nucleicacids.” J Gene Med 5(3): 232-45).

One specific approach in gene therapy uses cationic lipids. However,although many cationic lipids show excellent transfection activity incell culture, most do not perform well in the presence of serum, andonly a few are active in vivo. A dramatic change in size, surfacecharge, and lipid composition occurs when lipoplexes are exposed to theoverwhelming amount of negatively charged and often amphipatic proteinsand polysaccharides that are present in blood, mucus epithelial liningfluid, or tissue matrix. Once administered in vivo, lipoplexes tend tointeract with negatively charged blood components and form largeaggregates that could be absorbed onto the surface of circulating redblood cells, trapped in a thick mucus layer or embolized inmicrovasculatures, preventing them from reaching the intended targetcells in the distal location. Furthermore, toxicity related to genetransfer by lipoplexes has been observed. Symptoms include inter aliainduction of inflammatory cyokines. In humans, various degrees ofadverse inflammatory reactions, including flu-like symptoms were notedamong subjects who received lipoplexes. Accordingly, it appearsquestionable, as to whether lipoplexes can be safely used in humans atall.

One further, more promising approach in gene therapy utilizes cationicpolymers. Cationic polymers turned out to be efficient in transfectionof nucleic acids, as they can tightly complex and condense a negativelycharged nucleic acid. Thus, a number of cationic polymers have beenexplored as carriers for in vitro and in vivo gene delivery. Theseinclude polyethylenimine (PEI), polyamidoamine and polypropylaminedendrimers, polyallylamine, cationic dextran, chitosan, cationicproteins and cationic peptides. Although most cationic polymers sharethe function of condensing DNA into small particles and facilitatingcellular uptake via endocytosis through charge-charge interaction withanionic sites on cell surfaces, their transfection activity and toxicitydiffer dramatically. Interestingly, cationic polymers exhibit bettertransfection efficiency with rising molecular weight due to strongercomplexation of the negatively charged nucleic acid cargo. However, arising molecular weight also leads to a rising toxicity of the cationicpolymer. PEI is perhaps the most active and most studied polymer forgene delivery, but its main drawback as a transfection reagent relatesto its non-biodegradable nature and toxicity. Furthermore, even thoughpolyplexes formed by high molecular weight polymers exhibit improvedstability under physiological conditions, data have indicated that suchpolymers can hinder vector unpacking. For example, poly (L-lysine) (PLL)of 19 and 36 residues was shown to dissociate from DNA more rapidly thanPLL of 180 residues resulting in significantly enhanced short-term geneexpression. A minimum length of six to eight cationic amino acids isrequired to compact DNA into structures active in receptor-mediated genedelivery. However, polyplexes formed with short polycations are unstableunder physiological conditions and typically aggregate rapidly inphysiological salt solutions. To overcome this negative impact, Read etal. (see Read, M. L., K. H. Bremner, et al. (2003). “Vectors based onreducible polycations facilitate intracellular release of nucleicacids.” J Gene Med 5(3): 232-45; and Read, M. L., S. Singh, et al.(2005). “A versatile reducible polycation-based system for efficientdelivery of a broad range of nucleic acids.” Nucleic Acids Res 33(9):e86) developed a new type of synthetic vector based on a linearreducible polycation (RPC) prepared by oxidative polycondensation of thepeptide Cys-Lys₁₀-Cys that can be cleaved by the intracellularenvironment to facilitate release of nucleic acids. They could show thatpolyplexes formed by RPC are destabilised by reducing conditionsenabling efficient release of DNA and mRNA. Cleavage of the RPC alsoreduced toxicity of the polycation to levels comparable with lowmolecular weight peptides. The disadvantage of this approach of Read etal. (2003, supra) was that the endosomolytic agent chloroquine or thecationic lipid DOTAP was additionally necessary to enhance transfectionefficiency to adequate levels. As a consequence Read et al. (2005,supra) included histidine residues in the RPCs which have a knownendosomal buffering capacity. They could show that histidine-rich RPCscan be cleaved by the intracellular reducing environment enablingefficient cytoplasmic delivery of a broad range of nucleic acids,including plasmid DNA, mRNA and siRNA molecules without the requirementfor the endosomolytic agent chloroquine.

Unfortunately; Read et al. (2005, supra) did not assess whetherhistidine-rich RPCs can be directly used for in vivo applications. Intheir study, transfections were performed in the absence of serum toavoid masking the ability of histidine residues to enhance gene transferthat may have arisen from binding of serum proteins to polyplexesrestricting cellular uptake. Preliminary experiments indicate that thetransfection properties of histidine-rich RPC polyplexes can be affectedby the presence of serum proteins with a 50% decrease in GFP-positivecells observed in 10% FCS (fetal calf serum). For in vivo applicationthey propose modifications with the hydrophilic polymer poly-[N-(2hydroxy-propyl)methacrylamide]. Unfortunately, Read et al. (2005, supra)did not prevent aggregation of polyplexes and binding of polycationicproteins to serum proteins. Furthermore, due to the large excess ofpolymer, which is characterized by the high N/P ratio, strong cationiccomplexes are formed when complexing the nucleic acid, which are only oflimited use in vivo due to their strong tendency of salt inducedagglomeration and interactions with serum contents (opsonization).Additionally, these complexes may excite an acute immune response, whenused for purposes of gene therapy. Read et al. (2003, supra) did alsonot provide in vivo data for the RPC based complexes shown in thepublication. It has also turned out that these strong cationic RPC basedcomplexes are completely inactive subsequent to local administrationinto the dermis. Furthermore Read et al. (2005, supra) used stringentoxidation conditions (30% DMSO) to induce the generation of highmolecular polymers with as long as possible chain lengths (“step-growthpolymerization”) to ensure complete complexation of the nucleic acidcargo.

In an approach similar to Read et al. McKenzie et al. (McKenzie, D. L.,K. Y. Kwok, et al. (2000), J Biol Chem 275(14): 9970-7., McKenzie, D.L., E. Smiley, et al. (2000), Bioconjug Chem 11(6): 901-9, and U.S. Pat.No. 6,770,740 B1) developed self-crosslinking peptides as gene deliveryagents by inserting multiple cysteines into short synthetic peptides forthe purpose of decreasing toxicity as observed with high-molecularpolycations. For complexation of DNA they mixed the self-crosslinkingpeptides with DNA to induce interpeptide disulfide bonds concurrently tocomplexation of the DNA cargo. For in vivo gene delivery approaches theypropose the derivatization of the self-crosslinking peptides with astealthing (e.g. polyethylene glycol) or targeting agent operativelyattached to the peptide at a site distal from each terminus. In afurther approach the same authors developed for the purpose of maskingDNA peptide condensates and thereby reducing interaction with bloodcomponents, the derivatization of the non crosslinking cationic peptideCWK₁₈ with polyethylene glycol by reducible or non-reducible linkages(Kwok, K. Y., D. L. McKenzie, et al. (1999). “Formulation of highlysoluble poly(ethylene glycol)-peptide DNA condensates.” J Pharm Sci88(10): 996-1003).

Summarizing the above, the present prior art as exemplified abovesuffers from various disadvantages. One particular disadvantage of theself-crosslinking peptides as described by Read et al. (2003, supra) orMcKenzie et al. (2000 I and II, supra and U.S. Pat. No. 6,770,740 B1)concerns the high positive charge on the surface of the particlesformed. Due to the high positive charge the particles exhibit a highinstability towards agglomeration when subjecting these particles invivo to raised salt concentrations. Such salt concentrations, however,typically occur in vivo in cells or extracellular media. Furthermore,high positively charged complexes show a strong tendency ofopsonization. This leads to an enhanced uptake by macrophages andfurthermore to a fast inactivation of the complex due to degradation.Particularly the uptake of these complexes by cells of the immune systemin general leads to a downstream stimulation of different cytokines.This unspecific activation of the innate immune system, however,represents a severe disadvantage of these systems and should be avoided,particularly for the purpose of several aspects of gene therapy, wherean acute immune response (cytokine storm) is strictly to be avoided.Additionally, in biological systems positively charged complexes caneasily be bound or immobilized by negatively charged components of theextracellular matrix or the serum. Also, the nucleic acids in thecomplex may be released too early, leading to reduced efficiency of thetransfer and half life of the complexes in vivo. Furthermore, areversible derivatization of carriers with a stealthing agent beingadvantageous for in vivo gene delivery, such as polyethylene glycol(PEG), was only possible for peptide monomers but not forself-crosslinking peptides or rather for a polymeric carrier with adefined polymer chain length. In particular, such a reversiblederivatization was not possible at the terminal ends of the crosslinkedcationic peptide carrier. Additionally, in the prior art onlyhigh-molecular polymers with long polymer chains or with an undefinedpolymer chain length consisting of self-crosslinking peptides weredescribed, which unfortunately compact their cargo to such an extentthat cargo release in the cell is limited. The extremely undefinedpolymer chain length is further problematic regarding approvement of amedicament based on RPC. One precondition for an approvement of amedicament is that every preparation of the medicament has always thesame composition, the same structure and the same properties. Thiscannot be ensured for complexes based on RPC's from the prior art.Furthermore the RPC-based polymers or complexes provided in the priorart are difficult to characterize due to their undefined structure orpolymer chain length. But characterization of the resulting complex orof the polymeric carrier is absolutly necessessary for the approvementof a medicament.

In consequence, no feasible method or carrier has been presented untiltoday, which allows both compacting and stabilizing a nucleic acid forthe purposes of gene therapy and other therapeutic applications, whichshow a good transfection activity in combination with a good release ofthe nucleic acid cargo, particularly in vivo and low or even notoxicity, e.g. due to the combination of a reversible stealthing and areversible complexation of the nucleic acid by self-crosslinkingpolymers. Accordingly, there is still an intensive need in the art toprovide carriers for the purpose of gene transfer, which are on the onehand side stable enough to carry their cargo to the target before theyare metabolically cleaved, and which on the other hand side can becleared from the tissue before they can accumulate and reach toxiclevels.

The object underlying the present invention is therefore to provide acarrier or a complexing agent, particularly for the transfection ofnucleic acids for the purposes of gene therapy or other therapeuticapplications, which is capable to compact nucleic acids, preferablycoding DNA or coding RNA, such as mRNA, and which allows efficienttransfection of the nucleic acid into different cell lines in vitro butalso transfection in vivo. As uptake by cells occurs via the endosomalroute, such a carrier or a complexing agent shall also allow or providefor efficient release of the nucleic acid. Additionally, it ispreferred, that the inventive polymeric carrier cargo complex formed bythe nucleic acid cargo and such carrier or a complexing agent showsimproved resistance to agglomeration. It should preferably also conferenhanced stability to the nucleic acid cargo with respect to serumcontaining media. Most importantly, in vivo activity shall be obtained,thereby suppressing at least in part an acute immune reaction.Furthermore a reproducible production and a characterization of thecarrier should be assured.

This object is solved by the subject matter of the present invention,preferably by the subject matter of the attached claims. Particularly,according to the first embodiment of the present invention the aboveobject is solved by a polymeric carrier molecule according to genericformula (I):

L-P¹—S—[S—P²—S]_(n)—S—P³-L

wherein,

-   P¹ and P³ are different or identical to each other and represent a    linear or branched hydrophilic polymer chain, each P¹ and P³    exhibiting at least one —SH-moiety, capable to form a disulfide    linkage upon condensation with component P², or alternatively with    (AA)_(x), or [(AA)_(x)]_(z) if such components are used as a linker    between P¹ and P² or P³ and P²) and/or with further components (e.g.    (AA)_(x), [(AA)_(x)]_(z) or L), the linear or branched hydrophilic    polymer chain selected independent from each other from polyethylene    glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide,    poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl    L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),    hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the    hydrophilic polymer chain exhibits a molecular weight of about 1 kDa    to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or more    preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to about    25 kDa or 5 kDa to about 10 kDa;-   P² is a cationic or polycationic peptide or protein, and preferably    having a length of about 3 to about 100 amino acids, more preferably    having a length of about 3 to about 50 amino acids, even more    preferably having a length of about 3 to about 25 amino acids, e.g.    a length of about 3 to 10, 5 to 15, 10 to 20 or 15 to 25 amino    acids, more preferably a length of about 5 to about 20 and even more    preferably a length of about 10 to about 20;    -   is a cationic or polycationic polymer, typically having a        molecular weight of about 0.5 kDa to about 30 kDa, including a        molecular weight of about 1 kDa to about 20 kDa, even more        preferably of about 1.5 kDa to about 10 kDa, or having a        molecular weight of about 0.5 kDa to about 100 kDa, including a        molecular weight of about 10 kDa to about 50 kDa, even more        preferably of about 10 kDa to about 30 kDa;    -   each P² exhibiting at least two —SH-moieties, capable to form a        disulfide linkage upon condensation with further components P²        or component(s) P¹ and/or P³ or alternatively with further        components (e.g. (AA)_(x), or [(AA)_(x)]_(z))-   —S—S— is a (reversible) disulfide bond (the brackets are omitted for    better readability), wherein S preferably represents sulphur or a    —SH carrying moiety, which has formed a (reversible) disulfide bond.    The (reversible) disulfide bond is preferably formed by condensation    of —SH-moieties of either components P¹ and P², P² and P², or P² and    P³, or optionally of further components as defined herein (e.g. L,    (AA)_(x), [(AA)_(x)]_(z), etc.); The —SH-moiety may be part of the    structure of these components or added by a modification as defined    below;-   L is an optional ligand, which may be present or not, and may be    selected independent from the other from RGD, Transferrin, Folate, a    signal peptide or signal sequence, a localization signal or    sequence, a nuclear localization signal or sequence (NLS), an    antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand    of a receptor (e.g. cytokines, hormones, growth factors etc.), small    molecules (e.g. carbohydrates like mannose or galctose or synthetic    ligands), small molecule agonists, inhibitors or antagonists of    receptors (e.g. RGD peptidomimetic analogues) etc.;-   n is an integer, typically selected from a range of about 1 to 50,    preferably from a range of about 1, 2 or 3 to 30, more preferably    from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1,    2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or    a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of    about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a    range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of    about 6 to 11 or 7 to 10. Most preferably, n is in a range of about    1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3,    or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of    about 1, 2, or 3 to 7.

The inventive polymeric carrier molecule according to generic formula(I) is prepared by a new synthesis strategy and advantageously allows todefine the length of the polymer chain and to combine desired propertiesof different (short) polymers in one polymer, e.g. to efficientlycompact nucleic acids for the purpose of efficient transfection ofnucleic acids for the purposes of gene therapy or other therapeuticapplications without loss of activity, particularly efficienttransfection of a nucleic acid into different cell lines in vitro butalso transfection in vivo. The inventive polymeric carrier molecule isfurthermore not toxic to cells and provides for efficient release of itsnucleic acid cargo. Finally, it shows improved resistance toagglomeration due to the reversible addition of hydrophilic polymerchains (e.g. PEG-monomers) particularly to the terminal ends of theinventive polymeric carrier molecule according to generic formula (I),which additionally confers enhanced stability of the nucleic acid cargowith respect to serum containing media and prevents recognition of thepolymeric carrier cargo complex by the immune system.

Even more advantageously, the inventive polymeric carrier moleculeaccording to generic formula (I) allows to considerably vary its peptideor polymeric content and thus to modulate its biophysical/biochemicalproperties, particularly the cationic properties of component[S—P²—S]_(n), quite easily and fast, e.g. by incorporating as componentsP² the same or different cationic peptide(s), protein(s) or polymer(s)and optionally adding other components, e.g. amino acid component(s)(AA)_(x), into the repetitive component [S—P²—S] to form a modifiedrepetitive component such as {[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)} as a coremotif of the inventive polymeric carrier (wherein a+b=n, see below).Even though consisting of quite small non-toxic monomer units theinventive polymeric carrier molecule forms a cationic binding sequencewith a defined chain length providing a strong condensation of thenucleic acid cargo and complex stability. Under the reducing conditionsof the cytosole (e.g. cytosolic GSH), the complex is rapidly degradedinto its monomers, which are further degraded (e.g. oligopeptides) orsecreted (e.g. PEG). This supports deliberation of the nucleic acidcargo in the cytosol. Due to degradation into small oligopeptides in thecytosole, no toxicity is observed as known for high-molecularoligopeptides, e.g. from high-molecular oligoarginine. The PEG-“coating”also allows to somehow “coat” the polymeric carrier with a hydrophiliccoating at its terminal ends, which prevents salt-mediated agglomerationand undesired interactions with serum contents. In the cytosole, this“coating” is easily removed under the reducing conditions of the cell.Also, this effect promotes deliberation of the nucleic acid cargo in thecytosol.

As defined above, ligands (L), may be optionally used in the inventivepolymeric carrier molecule according to generic formula (I), e.g. fordirection of the inventive carrier polymer and its complexed nucleicacid into specific cells. They may be selected independent from theother from RGD, Transferrin, Folate, a signal peptide or signalsequence, a localization signal or sequence, a nuclear localizationsignal or sequence (NLS), an antibody, a cell penetrating peptide, (e.g.TAT), a ligand of a receptor (e.g. cytokines, hormones, growth factorsetc.), small molecules (e.g. carbohydrates like mannose or galctose orsynthetic ligands), small molecule agonists, inhibitors or antagonistsof receptors (e.g. RGD peptidomimetic analogues) etc. Particularlypreferred in this context is mannose as ligand to target antigenpresenting cells which carries on their cell membrane mannose receptors.In a further preferred aspect of the first embodiment of the presentinvention galactose as optional ligand can be used to targethepatocytes. Such ligands may be attached to component P¹ and/or P³ byreversible disulfide bonds as defined below or by any other possiblechemical attachment, e.g. by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc.), by Michael addition (e.g. maleinimidemoieties, α,β unsatured carbonyls, etc.), by click chemistry (e.g.azides or alkines), by alkene/alkine methatesis (e.g. alkenes oralkines), imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.g.halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components.

In the context of formula (I) of the present invention components P¹ andP³ represent a linear or branched hydrophilic polymer chain, containingat least one —SH-moiety, each P¹ and P³ independently selected from eachother, e.g. from polyethylene glycol (PEG),poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethylphosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkylL-glutamine). P¹ and P³ may be identical or different to each other.Preferably, each of hydrophilic polymers P¹ and P³ exhibits a molecularweight of about 1 kDa to about 100 kDa, preferably of about 1 kDa toabout 75 kDa, more preferably of about 5 kDa to about 50 kDa, even morepreferably of about 5 kDa to about 25 kDa. Additionally, each ofhydrophilic polymers P¹ and P³ typically exhibits at least one—SH-moiety, wherein the at least one —SH-moiety is capable to form adisulfide linkage upon condensation with component P² or with component(AA)_(x), if used as linker between P¹ and P² or P³ and P² as definedbelow and optionally with a further component, e.g. L and/or (AA)_(x),e.g. if two or more —SH-moieties are contained. The followingsubformulas “P¹—S—S—P²” and “P²—S—S—P³” of generic formula (I) above(the brackets are omitted for better readability), wherein any of S, P¹and P³ are as defined herein, typically represent a situation, whereinone —SH-moiety of hydrophilic polymers P¹ and P³ was condensed with one—SH-moiety of component P² of generic formula (I) above, wherein bothsulphurs of these —SH-moieties form a disulfide bond S—S— as definedherein in formula (I). These —SH-moieties are typically provided by eachof the hydrophilic polymers P¹ and P³, e.g. via an internal cysteine orany further (modified) amino acid or compound which carries a —SHmoiety. Accordingly, the subformulas “P¹—S—S—P²” and “P²—S—S—P³” mayalso be written as “P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”, if the—SH-moiety is provided by a cysteine, wherein the term Cys-Cysrepresents two cysteines coupled via a disulfide bond, not via a peptidebond. In this case, the term “—S—S—” in these formulae may also bewritten as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, theterm “-Cys-Cys-” does not represent a peptide bond but a linkage of twocysteines via their —SH-moieties to form a disulfide bond. Accordingly,the term “-Cys-Cys-” also may be understood generally as“-(Cys-S)—(S-Cys)-”, wherein in this specific case S indicates thesulfur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and“—Cys-S” indicate a disulfide bond between a —SH containing moiety and acysteine, which may also be written as “—S—(S—Cys)” and “-(Cys-S)—S”.Alternatively, the hydrophilic polymers P¹ and P³ may be modified with a—SH moiety, preferably via a chemical reaction with a compound carryinga —SH moiety, such that each of the hydrophilic polymers P¹ and P³carries at least one such —SH moiety. Such a compound carrying a —SHmoiety may be e.g. an (additional) cysteine or any further (modified)amino acid, which carries a —SH moiety. Such a compound may also be anynon-amino compound or moiety, which contains or allows to introduce a—SH moiety into hydrophilic polymers P¹ and P³ as defined herein. Suchnon-amino compounds may be attached to the hydrophilic polymers P¹ andP³ of formula (I) according to the present invention via chemicalreactions or binding of compounds, e.g. by binding of a 3-thio propionicacid or thioimolane, by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc.), by Michael addition (e.g. maleinimidemoieties, α,β unsatured carbonyls, etc.), by click chemistry (e.g.azides or alkines), by alkene/alkine methatesis (e.g. alkenes oralkines), imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.g.halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components. Aparticularly preferred PEG derivate in this context isalpha-Methoxy-omega-mercapto poly(ethylene glycol). In each case, the—SH-moiety, e.g. of a cysteine or of any further (modified) amino acidor compound, may be present at the terminal ends or internally at anyposition of hydrophilic polymers P¹ and P³. As defined herein, each ofhydrophilic polymers P¹ and P³ typically exhibits at least one—SH-moiety preferably at one terminal end, but may also contain two oreven more —SH-moieties, which may be used to additionally attach furthercomponents as defined herein, e.g. a ligand, an amino acid component(AA)_(x), antibodies, cell penetrating peptides (e.g. TAT), etc.

According to a further preferred aspect of the first embodiment of thepresent invention, each of hydrophilic polymers P¹ and P³ may alsocontain at least one further functional moiety, which allows attachingfurther components as defined herein, e.g. a ligand, an amino acidcomponent (AA)_(x), etc. Such functional moieties may be selected fromfunctionalities which allow the attachment of further components, e.g.functionalities as defined herein, e.g. by amide formation (e.g.carboxylic acids, sulphonic acids, amines, etc.), by Michael addition(e.g. maleinimide moieties, α,β unsatured carbonyls, etc.), by clickchemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g.alkenes or alkines), imine or hydrozone formation (aldehydes or ketons,hydrazins, hydroxylamins, amines), complexation reactions (avidin,biotin, protein G) or components which allow S_(n)-type substitutionreactions (e.g. halogenalkans, thiols, alcohols, amines, hydrazines,hydrazides, sulphonic acid esters, oxyphosphonium salts) or otherchemical moieties which can be utilized in the attachment of furthercomponents.

Component P² in the context of formula (I) of the present inventionpreferably represents a cationic or polycationic peptide or protein oralternatively a cationic or polycationic polymer. Each component P²typically exhibits at least two —SH-moieties, capable to form adisulfide linkage upon condensation with further components P²,component(s) P¹ and/or P³ or alternatively with further components, e.g.amino acid components (AA)_(x). Component P² typically occurs within therepetitive component [—S—P²—S—]_(n) of formula (I) of the presentinvention. The term “cationic or polycationic” typically refers to acharged molecule, which is positively charged (cation) at a pH value ofabout 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferablyof a pH value of or below 9, of or below 8, of or below 7, mostpreferably at physiological pH values, e.g. about 7.3 to 7.4.Accordingly, a cationic or polycationic peptide or protein as componentP² or alternatively a cationic or polycationic polymer as component P²according to the present invention is positively charged underphysiological conditions, particularly under physiological saltconditions of the cell in viva

In the specific case that component P² of formula (I) of the presentinvention is a cationic or polycationic peptide or protein the cationicproperties of component [S—P²—S]_(n) or {[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)(as defined below) may be determined upon its content of cationic aminoacids in the entire component [S—P²—S]_(n) or{[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)}. Preferably, the content of cationicamino acids in component [S—P²—S]_(n) or{[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)} is at least 10%, 20%, or 30%,preferably at least 40%, more preferably at least 50%, 60% or 70%, butalso preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%,more preferably in the range of about 15% to 75%, even more preferablyin the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a rangeformed by any two of the afore mentioned values, provided, that thecontent of all amino acids, e.g. cationic, lipophilic, hydrophilic,aromatic and further amino acids, in the entire component [S—P²—S]_(n)or {[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)} is 100%.

In the specific case that component P² of formula (I) of the presentinvention is a cationic or polycationic polymer the cationic propertiesof component [S—P²—S]_(n) or {[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)} may bedetermined upon its content of cationic charges in the entire component[S—P²—S]_(n) or {[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)} when compared to theoverall charges of component [S—P²—S]_(n) or{[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)}. Preferably, the content of cationiccharges in component [S—P²—S]_(n) or {[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)}at a (physiological) pH as defined herein is at least 10%, 20%, or 30%,preferably at least 40%, more preferably at least 50%, 60% or 70%, butalso preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%,more preferably in the range of about 15% to 75%, even preferably in therange of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a range formedby any two of the afore mentioned values, provided, that the content ofall charges, e.g. positive and negative charges at a (physiological) pHas defined herein, in the entire component [S—P²—S]_(n) or{[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)} is 100%.

In the specific context of the inventive polymeric carrier cargo complexformed by the nucleic acid cargo and a polymeric carrier moleculeaccording to generic formula (I) L-P¹—S—[S—P²—S]_(n)—S—P³-L as definedherein (or according to any of its subformulas herein) it isparticularly preferred that at least 10% of all charges in the wholerepetitive component [S—P²—S]_(n) or {[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)}are cationic to allow complexation of the negatively charged nucleicacid cargo.

The cationic or polycationic peptide or protein as component P², or thecationic or polycationic polymer as component P², is preferably a linearmolecule, however, branched cationic or polycationic peptides orproteins as component P² or branched cationic or polycationic polymersas component P² may also be used.

Typically, component P², e.g. the cationic or polycationic peptide orprotein or the cationic or polycationic polymer as defined herein, islinked to its neighboring components, e.g. components P¹ and P³, and/oras a part of repetitive component [S—P²—S]_(n) to further components P²,via disulfide bonds (—S—S—) or to (AA)_(x) components as part of{[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)}. In this context, the sulfurs adjacentto component P² in the repetitive component [S—P²—S]_(n) and as definedin generic formula (I) L-P¹—S—[S—P²—S]_(n)—S—P³-L, necessary forproviding a disulfide bond, may be provided by component P² itself by a—SH moiety as defined herein or may be provided by modifying componentP² accordingly to exhibit a —SH moiety within the above definition ofrepetitive component [S—P²—S]. The —SH moieties for component P² arepreferably as defined herein for components P¹ and P³. If such—SH-moieties, necessary to form a disulfide bond (—S—S—) within theabove meaning, are provided by component P² itself this may occur e.g.by at least two cysteines or any further (modified) amino acids orchemical compounds, which carry a —SH moiety, already occurring withinthe amino acid sequence of component P² at whatever position of theamino acid sequence of component P². Alternatively, component P² may bemodified accordingly with a chemical compound, e.g. a cysteine or anyfurther (modified) amino acid or chemical compound, which carries a(free) —SH moiety. Thereby, component P² preferably carries at least two—SH-moieties, which sulphurs atoms are capable to form a disulfide bondupon condensation with a —SH-moiety of components P¹ or P³ as definedherein, or between a first component P² and a further component P², etc.Such —SH-moieties are preferably as defined herein. Preferably the atleast two SH-moieties are located at the terminal ends or near to theterminal ends of component P²

According to one specific aspect of the first embodiment of the presentinvention, component P² within repetitive component [S—P²—S]_(n) ofgeneric formula (I) above may comprise a cysteine as a —SH moiety. Inthis context, repetitive component [S—P²—S]_(n) may thus be written asfollows:

[Cys-P²-Cys]_(n)

wherein n and P² are as defined herein and each Cys provides for the—SH-moiety for the disulfide bond. Cys is the amino acid cysteine in itsthree letter code. (For illustrative purposes, in the presentdescription the disulfide bond —S—S— generally may also be written as-(Cys-S)—(S-Cys)-, wherein Cys-S represents a Cysteine with an naturallyoccurring —SH moiety, wherein this —SH moiety forms a disulfide bondwith a —SH moiety of a second cysteine. Accordingly, repetitivecomponent [Cys-P²-Cys]_(n) may also be written as[(S-Cys)-P²-(Cys-S)]_(n), which indicates that the —SH-moiety isprovided by a cysteine and the Cysteine itself provides for the sulfurof the disulfide bond.)

In the context of the entire formula (I) above, this specific aspect ofthe first embodiment thus may be defined as follows:

L-P¹—S-[Cys-P²-Cys]_(n)—S—P³-L

wherein L, P¹, P², P³ and n are as defined herein, S is sulphur and eachCys provides for one —SH-moiety for the disulfide bond.

In each case, the SH-moiety, e.g. of a cysteine or any further(modified) amino acid or further compound used for modification ofcomponent P², may be present in the cationic or polycationic peptide orprotein or cationic or polycationic polymer as component P², internallyor at one or both of its terminal ends, e.g. if a cationic orpolycationic peptide or protein is used as component P² at theN-terminal end or at the C-terminal end, at both these terminal ends,and/or internally at any position of the cationic or polycationicpeptide or protein as component P². Preferably, the —SH moiety may bepresent in component P² at least at one terminal end, more preferably atboth terminal ends, e.g. at the N-terminal end and/or at the C-terminalend, more preferably at both the N-terminal and the C-terminal end of acationic or polycationic peptide or protein as component P².

Due to its repetitive character component [S—P²—S]_(n) may represent asituation, wherein one of the at least two —SH-moieties of component P²was condensed with a —SH-moiety of a further component P² of genericformula (I) above, wherein both sulphurs of these —SH-moieties form adisulfide bond (—S—S—) between a first component P² and at least onefurther component P².

In this context, the number of repetitions of component P² in formula(I) according to the present invention is defined by integer n. n is aninteger, typically selected from a range of about 1 to 50, preferablyfrom a range of about 1, 2 or 3 to 30, more preferably from a range ofabout 1, 2, 3, 4, or 5 to 25, or a range of about 1, 2, 3, 4, or 5 to20, or a range of about 1, 2, 3, 4, or 5 to 15, or a range of about 1,2, 3, 4, or 5 to 10, including e.g. a range of about 3 to 20, 4 to 20, 5to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10to 15, or a range of about 6 to 11 or 7 to 10. If, for example, inrepetitive component [S—P²—S]_(n) integer n is 5, repetitive component[S—P²—S]_(n) preferably reads as follows:

[S—P²—S—S—P²—S—S—P²—S—S—P²—S—S—P²—S]

In the above example component P² occurs 5 times (preferably in a linearorder), wherein each component P² is linked to its neighbor component bya disulfide bond within the above definition of repetitive component[S—P²—S]_(n). Any of components P² may be the same or different fromeach other.

According to one particular aspect of this embodiment, component P²represents a cationic or polycationic peptide or protein having a lengthof about 3 to about 100 amino acids, more preferably having a length ofabout 3 to about 50 amino acids, even more preferably having a length ofabout 3 to about 25 amino acids, e.g. having a length of about 3 to 10,5 to 15, 10 to 20 or 15 to 25 amino acids, more preferably a length ofabout 5 to about 20 and even more preferably a length of about 10 toabout 20.

The cationic or polycationic peptide or protein as component P² may beany protein or peptide suitable for this purpose and exhibiting at leasttwo —SH-moieties, particular any cationic or polycationic peptide orprotein capable to complex a nucleic acid as defined according to thepresent invention, and thereby preferably condensing the nucleic acid.

Particularly preferred, cationic or polycationic peptides or proteins ascomponent P² exhibiting at least two —SH-moieties may be selected fromprotamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL),basic polypeptides, poly-arginine, cell penetrating peptides (CPPs),chimeric CPPs, such as Transportan, or MPG peptides, HIV-bindingpeptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines,members of the penetratin family, e.g. Penetratin, Antennapedia-derivedpeptides (particularly from Drosophila antennapedia), pAntp, plsl, etc.,antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1),pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Proline-richpeptides, Loligomere, Arginine-rich peptides, Calcitonin-peptides, FGF,Lactoferrin, histones, VP22 derived or analog peptides, HSV, VP22(Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpT620, prolin-rich peptides, lysine-rich peptides, Pep-1, L-oligomers,Calcitonin peptide(s), etc.

According to one particular preferred aspect of the first embodiment ofthe present invention, cationic or polycationic peptides or proteins ascomponent P² are selected from following cationic peptides having thefollowing total sum formula (II), preferably under the provison thatthey exhibit additionally at least two —SH-moieties:

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};  (formula (II))

wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 10% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14,provided, that the overall content of Xaa does not exceed 90% of allamino acids of the oligopeptide. Any of amino acids Arg, Lys, His, Ornand Xaa may be positioned at any place of the peptide. Particularlypreferred peptides of this formula are oligoarginines such as e.g. Arg7,Arg₈, Arg₉, Arg H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc.(SEQ ID NOs: 1-9).

According to a particular preferred aspect of the first embodiment,cationic or polycationic peptides or proteins as component P², havingthe empirical formula (II) as shown above and additionally exhibiting atleast two —SH-moieties, may be, without being restricted thereto,selected from the following subgroup of formulae:

(SEQ ID NOs: 2-3, 10-15) Arg₈, Arg₉, Arg₁₀, Arg₁₁, Arg₁₂,Arg₁₃, Arg₁₄, Arg₁₅; (SEQ ID NOs: 16-23)Lys₈, Lys₉, Lys₁₀, Lys₁₁, Lys₁₂, Lys₁₃, Lys₁₄, Lys₁₅;(SEQ ID NOs: 24-31) His₈, His₉, His₁₀, His₁₁, His₁₂,His₁₃, His₁₄, His₁₅; (SEQ ID NOs: 32-39)Orn₈, Orn₉, Orn₁₀, Orn₁₁, Orn₁₂,  Orn₁₃, Orn₁₄, Orn₁₅;

According to a further particularly preferred aspect of the firstembodiment, cationic or polycationic peptides or proteins as componentP², having the empirical formula (II) as shown above and additionallyexhibiting at least two —SH-moieties, may be, without being restrictedthereto, selected from the following subgroup of formulae, wherein thefollowing formulae (as with empirical formula (II)) do not specify anyamino acid order, but are intended to reflect empirical formulae byexclusively specifying the (number of) amino acids as components of therespective peptide. Accordingly, as an example, empirical formulaArg₍₇₋₁₄₎Lys₁ is intended to mean that peptides falling under thisformula contain 7 to 14 Arg residues and 1 Lys residue of whatsoeverorder. If the peptides contain 7 Arg residues and 1 Lys residue, allvariants having 7 Arg residues and 1 Lys residue are encompassed. TheLys residue may therefore be positioned anywhere in the e.g. 8 aminoacid long sequence composed of 7 Arg and 1 Lys residues. The subgrouppreferably comprises:

Arg₍₇₋₁₄₎Lys₁, Arg₍₇₋₁₄₎His₁, Arg₍₇₋₁₄₎Orn₁, Lys₍₇₋₁₄₎His,Lys₍₇₋₁₄₎Orn₁, His₍₇₋₁₄₎Orn₁;Arg₍₆₋₁₃₎Lys₂, Arg₍₆₋₁₃₎His₂, Arg₍₆₋₁₃₎Orn₂, Lys₍₆₋₁₃₎His₂,Lys₍₆₋₁₃₎Orn₂, His₍₆₋₁₃₎Orn₂;Arg₍₅₋₁₂₎Lys₃, Arg₍₅₋₁₂₎His₃, Arg₍₅₋₁₂₎Orn₃, Lys₍₅₋₁₂₎His₃,Lys₍₅₋₁₂₎Orn₃, His₍₅₋₁₂₎Orn₃;Arg₍₄₋₁₁₎Lys₄, Arg₍₄₋₁₁₎His₄, Arg₍₄₋₁₁₎Orn₄, Lys₍₄₋₁₁₎His₄,Lys₍₄₋₁₁₎Orn₄, His₄₋₁₁₎Orn₄;Arg₍₃₋₁₀₎Lys₅, Arg₍₃₋₁₀₎His₅, Arg₍₃₋₁₀₎Orn₅, Lys₍₃₋₁₀₎His₅,Lys₍₃₋₁₀₎Orn₅, His₍₃₋₁₀₎Orn₅;Arg₍₂₋₉₎Lys₆, Arg₍₂₋₉₎His₆, Arg₍₂₋₉₎Orn₆, Lys₍₂₋₉₎His₆, Lys₍₂₋₉₎Orn₆,His₍₂₋₉₎Orn₆;Arg₍₁₋₈₎Lys₇, Arg₍₁₋₈₎His₇, Arg₍₁₋₈₎Orn₇, Lys₍₁₋₈₎His₇, Lys₍₁₋₈₎Orn₇,His₍₁₋₈₎Orn₇;Arg₍₆₋₁₃₎Lys₁His₁, Arg₍₆₋₁₃₎Lys, Orn₁, Arg₍₆₋₁₃₎His₁Orn₁,Arg₁Lys₍₆₋₁₃₎His₁, Arg₁Lys₍₆₋₁₃₎Orn₁, Lys₍₆₋₁₃₎His₁Orn₁,Arg₁Lys₁His₍₆₋₁₃₎, Arg₁His₍₆₋₁₃₎Orn₁, Lys₁His₍₆₋₁₃₎Orn₁;Arg₍₅₋₁₂₎Lys₂His₁, Arg₍₅₋₁₂₎Lys₁His₂, Arg₍₅₋₁₂₎Lys₂Orn₁,Arg₍₅₋₁₂₎Lys₁Orn₂, Arg₍₅₋₁₂₎His₂Orn₁, Arg₍₅₋₁₂₎His₁Orn₂,Arg₂Lys₍₅₋₁₂₎His₁, Arg₁Lys₍₅₋₁₂₎His₂, Arg₂Lys₍₅₋₁₂₎Orn₁,Arg₁Lys₍₅₋₁₂₎Orn₂, Lys₍₅₋₁₂₎His₂Orn₁, Lys₍₅₋₁₂₎His₁Orn₂,Arg₂Lys₁His₍₅₋₁₂₎, Arg₁Lys₂His₍₅₋₁₂₎, Arg₂His₍₅₋₁₂₎Orn₁,Arg₁His₍₅₋₁₂₎Orn₂, Lys₂His₍₅₋₁₂₎Orn₁, Lys₁His₍₅₋₁₂₎Orn₂;Arg₍₄₋₁₁₎Lys₃His₁, Arg₍₄₋₁₁₎Lys₂His₂, Arg₍₄₋₁₁₎Lys₁His₃,Arg₍₄₋₁₁₎Lys₃Orn₁, Arg₍₄₋₁₁₎Lys₂Orn₂, Arg₍₄₋₁₁₎Lys₁Orn₃,Arg₍₄₋₁₁₎His₃Orn₁, Arg₍₄₋₁₁₎His₂Orn₂, Arg₍₄₋₁₁₎His₁Orn₃,Arg₃Lys₍₄₋₁₁₎His₁, Arg₂Lys₍₄₋₁₁₎His₂, Arg₁Lys₍₄₋₁₁₎His₃,Arg₃Lys₍₄₋₁₁₎Orn₁, Arg₂Lys₍₄₋₁₁₎Orn₂, Arg₁Lys₍₄₋₁₁₎Orn₃,Lys₍₄₋₁₁₎His₃Orn₁, Lys₍₄₋₁₁₎His₂Orn₂, Lys₍₄₋₁₁₎His₁Orn₃,Arg₃Lys₁His₍₄₋₁₁₎, Arg₂Lys₂His₍₄₋₁₁₎, Arg₁Lys₃His₍₄₋₁₁₎,Arg₃His₍₄₋₁₁₎Orn₁, Arg₂His₍₄₋₁₁₎Orn₂, Arg₁His₍₄₋₁₁₎Orn₃,Lys₃His₍₄₋₁₁₎Orn₁, Lys₂His₍₄₋₁₁₎Orn₂, Lys₁His₍₄₋₁₁₎Orn₃;Arg₍₃₋₁₀₎Lys₄His₁, Arg₍₃₋₁₀₎Lys₃His₂, Arg₍₃₋₁₀₎Lys₂His₃,Arg₍₃₋₁₀₎Lys₁His₄, Arg₍₃₋₁₀₎Lys₄Orn₁, Arg₍₃₋₁₀₎Lys₃Orn₂,Arg₍₃₋₁₀₎Lys₂Orn₃, Arg₍₃₋₁₀₎Lys₁Orn₄, Arg₍₃₋₁₀₎His₄Orn₁,Arg₍₃₋₁₀₎His₃Orn₂, Arg₍₃₋₁₀₎His₂Orn₃, Arg₍₃₋₁₀₎His₁Orn₄,Arg₄Lys₍₃₋₁₀₎His₁, Arg₃Lys₍₃₋₁₀₎His₂, Arg₂Lys₍₃₋₁₀₎His₃,Arg₁Lys₍₃₋₁₀₎His₄, Arg₄Lys₍₃₋₁₀₎Orn₁, Arg₃Lys₍₃₋₁₀₎Orn₂,Arg₂Lys₍₃₋₁₀₎Orn₃, Arg₁Lys₍₃₋₁₀₎Orn₄, Lys₍₃₋₁₀₎His₄Orn₁,Lys₍₃₋₁₀₎His₃Orn₂, Lys₍₃₋₁₀₎His₂Orn₃, Lys₍₃₋₁₀₎His₁Orn₄,Arg₄Lys₁His₍₃₋₁₀₎, Arg₃Lys₂His₍₃₋₁₀₎, Arg₂Lys₃His₍₃₋₁₀₎,Arg₁Lys₄His₍₃₋₁₀₎, Arg₄His₍₃₋₁₀₎Orn₁, Arg₃His₍₃₋₁₀₎Orn₂,Arg₂His₍₃₋₁₀₎Orn₃, Arg₁His₍₃₋₁₀₎Orn₄, Lys₄His₍₃₋₁₀₎Orn₁,Lys₃His₍₃₋₁₀₎Orn₂, Lys₂His₍₃₋₁₀₎Orn₃, Lys₁His₍₃₋₁₀₎Orn₄;Arg₍₂₋₉₎Lys₅His₁, Arg₍₂₋₉₎Lys₄His₂, Arg₍₂₋₉₎Lys₃His₃, Arg₍₂₋₉₎Lys₂His₄,Arg₍₂₋₉₎Lys₁His₅, Arg₍₂₋₉₎Lys₅Orn₁, Arg₍₂₋₉₎Lys₄Orn₂, Arg₍₂₋₉₎Lys₃Orn₃,Arg₍₂₋₉₎Lys₂Orn₄, Arg₍₂₋₉₎Lys₁Orn₅, Arg₍₂₋₉₎His₅Orn₁, Arg₍₂₋₉₎His₄Orn₂,Arg₍₂₋₉₎His₃Orn₃, Arg₍₂₋₉₎His₂Orn₄, Arg₍₂₋₉₎His₁Orn₅, Arg₅Lys₍₂₋₉₎His,Arg₄Lys₍₂₋₉₎His₂, Arg₃Lys₍₂₋₉₎His₃, Arg₂Lys₍₂₋₉₎His₄, Arg₁Lys₍₂₋₉₎His₅,Arg₃Lys₍₂₋₉₎Orn₁, Arg₄Lys₍₂₋₉₎Orn₂, Arg₃Lys₍₂₋₉₎Orn₃, Arg₂Lys₍₂₋₉₎Orn₄,Arg₁Lys₍₂₋₉₎Orn₅, Lys₍₂₋₉₎His₅Orn, Lys₍₂₋₉₎His₄Orn₂, Lys₍₂₋₉₎His₃Orn₃,Lys₍₂₋₉₎His₂Orn₄, Lys₍₂₋₉₎His₁Orn₅, Arg₅Lys₁His₍₂₋₉₎, Arg₄Lys₂His₍₂₋₉₎,Arg₃Lys₃His₍₂₋₉₎, Arg₂Lys₄His₍₂₋₉₎, Arg₁Lys₅His₍₂₋₉₎, Arg₅His₍₂₋₉₎Orn₁,Arg₄His₍₂₋₉₎Orn₂, Arg₃His₍₂₋₉₎Orn₃, Arg₂His₍₂₋₉₎Orn₄, Arg₁His₍₂₋₉₎Orn₅,Lys₅His₍₂₋₉₎Orn₁, Lys₄His₍₂₋₉₎Orn₂, Lys₃His₍₂₋₉₎Orn₃, Lys₂His₍₂₋₉₎Orn₄,Lys₁His₍₂₋₉₎Orn₅;Arg₍₁₋₈₎Lys₆His₁, Arg₍₁₋₈₎Lys₅His₂, Arg₍₁₋₈₎Lys₄His₃, Arg₍₁₋₈₎Lys₃His₄,Arg₍₁₋₈₎Lys₂His₅, Arg₍₅₋₈₎Lys₁His₆, Arg₍₁₋₈₎Lys₆Orn₁, Arg₍₁₋₈₎Lys₅Orn₂,Arg₍₁₋₈₎Lys₄Orn₃, Arg₍₁₋₈₎Lys₃Orn₄, Arg₍₁₋₈₎Lys₂Orn₅, Arg₍₁₋₈₎Lys₁Orn₆,Arg₍₁₋₈₎His₆Orn₁, Arg₍₁₋₈₎His₅Orn₂, Arg₍₁₋₈₎His₄Orn₃, Arg₍₁₋₈₎His₃Orn₄,Arg₍₁₋₈₎His₂Orn₅, Arg₍₁₋₈₎His₁Orn₆, Arg₆Lys₍₁₋₈₎His₁, Arg₅Lys₍₁₋₈₎His₂,Arg₄Lys₍₁₋₈₎His₃, Arg₃Lys₍₁₋₈₎His₄, Arg₂Lys₍₁₋₈₎His₅, Arg₁Lys₍₁₋₈₎His₆,Arg₆Lys₍₁₋₈₎Orn₁, Arg₅Lys₍₁₋₈₎Orn₂, Arg₄Lys₍₁₋₈₎Orn₃, Arg₃Lys₍₁₋₈₎Orn₄,Arg₂Lys₍₁₋₈₎Orn₅, Arg₁Lys₍₁₋₈₎Orn₆, Lys₍₁₋₈₎His₆Orn₁, Lys₍₁₋₈₎His₅Orn₂,Lys₍₁₋₈₎His₄Orn₃, Lys₍₁₋₈₎His₃Orn₄, Lys₍₁₋₈₎His₂Orn₅, Lys₍₁₋₈₎His₁Orn₆,Arg₆Lys₁His₍₁₋₈₎, Arg₅Lys₂His₍₁₋₈₎, Arg₄Lys₃His₍₁₋₈₎, Arg₃Lys₄His₍₁₋₈₎,Arg₂Lys₅His₍₁₋₈₎, Arg₁Lys₆His₍₁₋₈₎, Arg₆His₍₁₋₈₎Orn₁, Arg₅His₍₁₋₈₎Orn₂,Arg₄His₍₁₋₈₎Orn₃, Arg₃His₍₁₋₈₎Orn₄, Arg₂His₍₁₋₈₎Orn₅, Arg₁His₍₁₋₈₎Orn₆,Lys₆His₍₁₋₈₎Orn₁, Lys₅His₍₁₋₈₎Orn₂, Lys₄His₍₁₋₈₎Orn₃, Lys₃His₍₁₋₈₎Orn₄,Lys₂His₍₁₋₈₎Orn₄, Lys₁His₍₁₋₈₎Orn₆,Arg₍₅₋₁₂₎Lys₁His₁Orn₁, Arg₁Lys₍₅₋₁₂₎His₁Orn₁, Arg₁Lys₁His₍₅₋₁₂₎Orn₁,Arg₁Lys₁His₁Orn₍₅₋₁₂₎;Arg₍₄₋₁₁₎Lys₂His₁Orn₁, Arg₍₄₋₁₁₎Lys₁His₂Orn, Arg₍₄₋₁₁₎Lys₁His₁Orn₂,Arg₂Lys₍₄₋₁₁₎His₁Orn₁, Arg₁Lys₍₄₋₁₁₎His₂Orn₁, Arg₁Lys₍₄₋₁₁₎His₁Orn₂,Arg₂Lys₁His₍₄₋₁₁₎Orn₁, Arg₁Lys₂His₍₄₋₁₁₎Orn₁, Arg₁Lys₁His₍₄₋₁₁₎Orn₂,Arg₂Lys₁His₁Orn₍₄₋₁₁₎, Arg₁Lys₂His₁Orn₍₄₋₁₁₎, Arg₁Lys₁His₂Orn₍₄₋₁₁₎;Arg₍₃₋₁₀₎Lys₃His₁Orn₁, Arg₍₃₋₁₀₎Lys₂His₂Orn₁, Arg₍₃₋₁₀₎Lys₂His₁Orn₂,Arg₍₃₋₁₀₎Lys₁His₂Orn₂, Arg₍₃₋ ₁₀₎Lys₁His₁Orn₃, Arg₃Lys₍₃₋₁₀₎His₁Orn₁,Arg₂Lys₍₃₋₁₀₎His₂Orn₁, Arg₂Lys₍₃₋₁₀₎His₁Orn₂, Arg₁Lys₍₃₋₁₀₎His₂Orn₂,Arg₁Lys₍₃₋₁₀₎His₁Orn₃, Arg₃Lys₁His₍₃₋₁₀₎Orn₁, Arg₂Lys₂His₍₃₋₁₀₎Orn₁,Arg₂Lys₁His₍₃₋₁₀₎Orn₂, Arg₁Lys₂His₍₃₋₁₀₎Orn₂, Arg₁Lys₁His₍₃₋₁₀₎Orn₃,Arg₃Lys₁His₁Orn₍₃₋₁₀₎, Arg₂Lys₂His₁Orn₍₃₋₁₀₎, Arg₂Lys₁His₂Orn₍₃₋₁₀₎,Arg₁Lys₂His₂Orn₍₃₋₁₀₎, Arg₁Lys₁His₃Orn₍₃₋₁₀₎;Arg₍₂₋₉₎Lys₄His₁Orn₁, Arg₍₂₋₉₎Lys, His₄Orn₁, Arg₍₂₋₉₎Lys₁His₁Orn₄,Arg₍₂₋₉₎Lys₃His₂Orn₁, Arg₍₂₋₉Lys₃His₁Orn₂, Arg₍₂₋₉₎Lys₂His₃Orn₁,Arg₍₂₋₉₎Lys₂His₁Orn₃, Arg₍₂₋₉₎Lys₁His₂Orn₃, Arg₍₂₋₉₎Lys₁His₃Orn₂,Arg₍₂₋₉₎Lys₂His₂Orn₂, Arg₄Lys₍₂₋₉₎His₁Orn₁, Arg₁Lys₍₂₋₉₎His₄Orn₁,Arg₁Lys₍₂₋₉₎His₁Orn₄, Arg₃Lys₍₂₋₉₎His₂Orn₁, Arg₃Lys₍₂₋₉₎His₁Orn₂,Arg₂Lys₍₂₋₉₎His₃Orn₁, Arg₂Lys₍₂₋₉₎His₁Orn₃, Arg₁Lys₍₂₋₉₎His₂Orn₃,Arg₁Lys₍₂₋₉₎His₃Orn₂, Arg₂Lys₍₂₋₉₎His₂Orn₂, Arg₄Lys₁His₍₂₋₉₎Orn₁,Arg₁Lys₄His₍₂₋₉₎Orn₁, Arg₁Lys₁His₍₂₋₉₎Orn₄, Arg₃Lys₂His₍₂₋₉₎Orn₁,Arg₃Lys₁His₍₂₋₉₎Orn₂, Arg₂Lys₃His₍₂₋₉₎Orn₁, Arg₂Lys₁His₍₂₋₉₎Orn₃,Arg₁Lys₂His₍₂₋₉₎Orn₃, Arg₁Lys₃His₍₂₋₉₎Orn₂, Arg₂Lys₂His₍₂₋₉₎Orn₂,Arg₄Lys₁His₁Orn₍₂₋₉₎, Arg₁Lys₄His₁Orn₍₂₋₉₎, Arg₁Lys₁His₄Orn₍₂₋₉₎,Arg₃Lys₂His₁Orn₍₂₋₉₎, Arg₃Lys₁His₂Orn₍₂₋₉₎, Arg₂Lys₃His₁Orn₍₂₋₉₎,Arg₂Lys₁His₃Orn₍₂₋₉₎, Arg₁Lys₂His₃Orn₍₂₋₉₎, Arg₁Lys₃His₂Orn₍₂₋₉₎,Arg₂Lys₂His₂Orn₍₂₋₉₎;Arg₍₁₋₈₎Lys₅His₁Orn₁, Arg₍₁₋₈₎Lys₁His₅Orn₁, Arg₍₁₋₈₎Lys₁His₁Orn₅,Arg₍₁₋₈₎Lys₄His₂Orn, Arg₍₁₋₈₎Lys₂His₄Orn₁, Arg₍₁₋₈₎Lys₂His₁Orn₄,Arg₍₁₋₈₎Lys₁His₂Orn₄, Arg₍₁₋₈₎Lys₁His₄Orn₂, Arg₍₁₋₈₎Lys₄His₁Orn₂,Arg₍₁₋₈₎Lys₃His₃Orn₁, Arg₍₁₋₈₎Lys₃His₁Orn₃, Arg₍₁₋₈₎Lys₁His₃Orn₃,Arg₅Lys₍₁₋₈₎His₁Orn₁, Arg₁Lys₍₁₋₈₎His₅Orn, Arg₁Lys₍₁₋₈₎His₁Orn₅,Arg₄Lys₍₁₋₈₎His₂Orn₁, Arg₂Lys₍₁₋₈₎His₄Orn₁, Arg₂Lys₍₁₋₈₎His₁Orn₄,Arg₁Lys₍₁₋₈₎His₂Orn₄, Arg₁Lys₍₁₋₈₎His₄Orn₂, Arg₄Lys₍₁₋₈₎His₁Orn₂,Arg₃Lys₍₁₋₈₎His₃Orn₁, Arg₃Lys₍₁₋₈₎His₁Orn₃, Arg₁Lys₍₁₋₈₎His₃Orn₃,Arg₅Lys, His₍₁₋₈₎Orn₁, Arg₁Lys₅His₍₁₋₈₎Orn₁, Arg₁Lys₁His₍₁₋₈₎Orn₅,Arg₄Lys₂His₍₁₋₈₎Orn₁, Arg₂Lys₄His₍₁₋₈₎Orn₁, Arg₂Lys₁His₍₁₋₈₎Orn₄,Arg₁Lys₂His₍₁₋₈₎Orn₄, Arg₁Lys₄His₍₁₋₈₎Orn₂, Arg₄Lys₁His₍₁₋₈₎Orn₂,Arg₃Lys₃His₍₁₋₈₎Orn₁, Arg₃Lys₁His₍₁₋₈₎Orn₃, Arg₁Lys₃His₍₁₋₈₎Orn₃,Arg₅Lys, His₁Orn₍₁₋₈₎, Arg₁Lys₅His₁Orn₍₁₋₈₎, Arg₁Lys₁His₅Orn₍₁₋₈₎,Arg₄Lys₂His₁Orn₍₁₋₈₎, Arg₂Lys₄His₁Orn₍₁₋₈₎, Arg₂Lys₁His₄Orn₍₁₋₈₎,Arg₁Lys₂His₄Orn₍₁₋₈₎, Arg₁Lys₄His₂Orn₍₁₋₈₎, Arg₄Lys₁His₂Orn₍₁₋₈₎,Arg₃Lys₃His₁Orn₍₁₋₈₎, Arg₃Lys₁His₃Orn₍₁₋₈₎, Arg₁Lys₃His₃Orn₍₁₋₈₎;

According to another particular preferred aspect of the firstembodiment, cationic or polycationic peptides or proteins as componentP², having the empirical formula (II) as shown above, and additionallyexhibiting at least two —SH-moieties may be, without being restrictedthereto, selected from following formulae: Arg₈, Arg₉, Arg₁₀, Arg₁₁,Arg₁₂, Arg₁₃, Arg₁₄, Arg₁₅; Lys₈, Lys₉, Lys₁₀, Lys₁₁, Lys₁₂, Lys₁₃,Lys₁₄, Lys_(1s); His_(s), His₉, His₁₀, His₁₁, His₁₂, His₁₃, His₁₄,His₁₅; Orn₈, Orn₉, Orn₁₀, Orn₁₁, Orn₁₂, Orn₁₃, Orn₁₄, Orn₁₅; (SEQ IDNOs: 2-3, 10-39, see above).

According to a further particular preferred aspect of the firstembodiment, cationic or polycationic peptides or proteins as componentP², having the empirical formula (II) as shown above, and additionallyexhibiting at least two —SH-moieties may be, without being restrictedthereto, selected from the subgroup consisting of generic formulas Arg₉(also termed R₉₎, Arg₉His₃ (also termed R₉H₃), His₃Arg₉His₃ (also termedH₃R₉H₃), TyrSerSerArg₉SerSerTyr (also termed YSSR₉SSY),His₃Arg₉SerSerTyr (also termed H₃R₉SSY), (ArgLysHis)₄ (also termed(RKH)₄), Tyr(ArgLysHis)₂Arg (also termed Y(RKH)₂R); (SEQ ID NOs: 2, 5-9,40, see above). Even more preferably, these generic formulas are definedas follows:

According to a one further particular preferred aspect of the firstembodiment, the cationic or polycationic peptide or protein as componentP², when defined according to formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (II)) asshown above, and additionally exhibiting at least two —SH-moieties maybe, without being restricted thereto, selected from formula (IIa),preferably under the provision that at least one —SH-moiety is providedby a cysteine residue:

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Cys)_(x)}  (formula (IIa))

wherein (Arg)_(l);(Lys)_(n);(His)_(n);(Orn)_(o); and x are as definedherein,

Alternatively, the cationic or polycationic peptide or protein ascomponent P², when defined according to formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (II)) asshown above, and additionally exhibiting at least two —SH-moieties maybe, without being restricted thereto, selected from formula (IIa′),preferably under the provision that at least one —SH— moiety is providedby a cysteine residue:

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x);(Cys)_(y)}  (formula(IIa′))

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80and 81-90, provided that the overall content of Arg (Arginine), Lys(Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% ofall amino acids of the oligopeptide.

These aspects of the first embodiment of the present invention may applyto situations, wherein component P² is selected from a cationic orpolycationic peptide or protein according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (II)) asshown above, which comprises or has been modified with at least onecysteine as —SH moiety in the above meaning such that the cationic orpolycationic peptide as component P² carries at least one cysteine,which is capable to form a disulfide bond with other components offormula (I).

According to another particular preferred aspect of the firstembodiment, the cationic or polycationic peptide or protein as componentP², when defined according to formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (II)) asshown above, and preferably additionally exhibiting at least two—SH-moieties may be, without being restricted thereto, selected fromformula (IIb), preferably under the provision that the at least two—SH-moieties are provided by two terminal cysteine residues:

Cys{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys  (formula(IIb))

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) are as definedherein and form a core of amino acids according to (semiempirical)formula (II). Exemplary examples may comprise any of the above sequencesflanked by two Cys and following sequences:

(SEQ ID NOs: 41 to 72) Cys(Arg₈)Cys, Cys(Arg₉)Cys, Cys(Arg₁₀)Cys,Cys(Arg₁₁)Cys, Cys(Arg₁₂)Cys, Cys(Arg₁₃)Cys,Cys(Arg₁₄)Cys, Cys(Arg₁₅)Cys; Cys(Lys₈)Cys,Cys(Lys₉)Cys, Cys(Lys₁₀)Cys, Cys(Lys₁₁)Cys,Cys(Lys₁₂)Cys, Cys(Lys₁₃)Cys, Cys(Lys₁₄)Cys,Cys(Lys₁₅)Cys; Cys(His₈)Cys, Cys(His₉)Cys,Cys(His₁₀)Cys, Cys(His₁₁)Cys, Cys(His₁₂)Cys,Cys(His₁₃)Cys, Cys(His₁₄)Cys, Cys(His₁₅)Cys;Cys(Orn₈)Cys, Cys(Orn₉)Cys, Cys(Orn₁₀)Cys,Cys(Orn₁₁)Cys, Cys(Orn₁₂)Cys, Cys(Orn₁₃)Cys,Cys(Orn₁₄)Cys, Cys(Orn₁₅)Cys,more preferably following exemplary sequences (SEQ ID NOs: 73 to 84):

CysArg₉Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Cys-CysCysArg₉His₃Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-His-His-His-CysCysHis₃Arg₉His₃Cys: Cys-His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His- His-His-Cys CysTyrSerSerArg₉SerCys-Tyr-Ser-Ser-Arg-Arg-Arg- SerTyrCys: Arg-Arg-Arg-Arg-Arg-Arg-Ser-Ser-Tyr-Cys CysHis₃Arg₉SerSer Cys-His-His-His-Arg-Arg-Arg- TyrCys:Arg-Arg-Arg-Arg-Arg-Arg-Ser- Ser-Tyr-Cys Cys(ArgLysHis)₄Cys:Cys-Arg-Lys-His-Arg-Lys-His- Arg-Lys-His-Arg-Lys-His-CysCysTyr(ArgLysHis)₂ Cys-Tyr-Arg-Lys-His-Arg-Lys- ArgCys: His-Arg-CysCysHis₃Arg₉His₃Cys: Cys-His-His-His-Arg-Arg-Arg- Arg-His-His-His-CysCysHis₆Arg₉His₆Cys: Cys-His-His-His-His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-His-His-His-His-His- His-CysCysHis₃Arg₄His₃Cys: Cys-His-His-His-Arg-Arg-Arg- Arg-His-His-His-CysCysHis₆Arg₄His₆Cys Cys-His-His-His-His-His-His-Arg-Arg-Arg-Arg-His-His-His- His-His-His-Cys CysArg₁₂Cys:Cys-Arg-Arg-Arg-Arg-Arg Arg- Arg-Arg-Arg-Arg-Arg-Arg-Cys

This aspect of the first embodiment of the present invention may applyto situations, wherein the polycationic peptide or protein as componentP², e.g. when defined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (II)) asshown above, has been modified with at least two (terminal) cysteines as—SH moieties in the above meaning such that component P² carries atleast two (terminal) cysteines, which are capable to form a disulfidebond with other components of formula (I).

According to another aspect of the first embodiment, component P²represents a cationic or polycationic polymer, selected from e.g. anycationic polymer suitable in this context, provided that this cationicpolymer exhibits at least two —SH-moieties, which provide for adisulfide bond linking component P² with component P¹ or P³, or withfurther component(s) P² or amino acid components (AA)_(x). Thus,likewise as defined herein, component P² may occur as a repetitivecomponent as defined herein as represented by subformula [S—P³—S], or{[S—P²—S]_(a)/[S-(AA)_(x)-S]_(b)}, wherein the same or differentcationic or polycationic polymers P² may be used in said repetitivecomponent.

Preferably, component P² represents a cationic or polycationic polymer,typically exhibiting a molecular weight of about 0.5 kDa to about 100kDa, of about 1 kDa to about 75 kDa, of about 5 kDa to about 50 kDa, ofabout 5 kDa to about 30 kDa, or a molecular weight of about 10 kDa toabout 50 kDa, or of about 10 kDa to about 30 kDa, preferably of about0.5 kDa to about 30 kDa, more preferably of about 1 kDa to about 20 kDa,and even more preferably of about 1.5 kDa to about 10 kDa. Additionally,the cationic or polycationic polymer as component P² typically exhibitsat least two —SH moieties, which are capable to form a disulfide linkageupon condensation with either components P¹ or P³ or with othercomponents P² or amino acid components (AA)_(x′) as defined herein.

When component P² represents a cationic or polycationic polymer, such apolymer may be selected from acrylates, modified acrylates, such aspDMAEMA (poly(dimethylaminoethyl methylacrylate)), chitosanes,aziridines or 2-ethyl-2-oxazoline (forming oligo ethylenimines ormodifed oligoethylenimines), polymers obtained by reaction ofbisacrylates with amines forming oligo beta aminoesters or poly amidoamines, or other polymers like polyesters, polycarbonates, etc. Eachmolecule of these cationic or polycationic polymers typically exhibitsat least two —SH-moieties, wherein these at least two —SH-moieties maybe introduced into the cationic or polycationic polymer by chemicalmodifications, e.g. using imonothiolan, 3-thio propionic acid orintroduction of —SH-moieties containing amino acids, such as cystein,methionine or any further (modified) amino acid. Such —SH-moieties arepreferably as already defined above for components P¹, P² or P³.

Component P² of formula (I) of the present invention preferably occursas repetitive component [—S—P²—S—]_(n). Such a repetitive component[S—P²—S]_(n) may be prepared using at least one or even more of the sameor different of the above defined components P² and polymerizing same ina polymerization condensation reaction via their —SH-moieties.

According to one specific aspect of the first embodiment, such arepetitive component [S—P²—S]_(n) may be prepared using at least one oreven more of the same or different of the above defined cationic orpolycationic peptides or proteins, and polymerizing same in apolymerization condensation reaction via their —SH-moieties.Accordingly, such a repetitive component [S—P²—S]_(n) contains a numberof at least one or even more of the same or different of the abovedefined cationic or polycationic proteins or peptides determined byinteger n.

According to another specific aspect of the first embodiment, such arepetitive component [S—P²—S]_(n) may be prepared using at least one oreven more of the same or different of the above defined cationic orpolycationic polymers, and polymerizing same in a polymerizationcondensation reaction via their —SH-moieties. Accordingly, such arepetitive component [S—P²—S]_(n) contains a number of at least one oreven more of the same or different of the above defined cationic orpolycationic polymers determined by integer n.

According to a further specific aspect of the first embodiment, such arepetitive component [S—P²—S]_(n) may be prepared using at least one oreven more of the same or different of the above defined cationic orpolycationic polymers and at least one or even more of the same ordifferent of the above defined cationic or polycationic proteins orpeptides, and polymerizing same in a polymerization condensationreaction via their —SH-moieties. Accordingly, such a repetitivecomponent [S—P²—S]_(n) contains a number of at least one or even more ofthe same or different of the above defined cationic or polycationicpolymers and at least one or even more of the same or different of theabove defined cationic or polycationic proteins or peptides, bothtogether determined by integer n.

According to a further aspect of the first embodiment, the inventivepolymeric carrier according to formula (I) above, may comprise at leastone amino acid component (AA)_(x), wherein AA is preferably an aminoacid as defined in the following, which, when occurring as amino acidcomponent (AA)_(x), allows to (substantially) modify thebiophysical/biochemical properties of the inventive polymeric carrieraccording to formula (I) as defined herein. According to the presentinvention, the number of amino acids in such an amino acid component(AA)_(x) (repetitions) is defined by x. In the above context, x ispreferably an integer and may be selected from a range of about 1 to100, preferably from a range of about 1 to 50, more preferably 1 to 30,and even more preferably selected from a number comprising 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-30, e.g. from a range of about 1to 30, from a range of about 1 to 15, or from a number comprising 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be selected from arange formed by any two of the afore mentioned values.

Such components (AA)_(x) may be contained in every parts of theinventive polymeric carrier according to formula (I) above and thereforemay be attached to all components of the inventive polymeric carrieraccording to formula (I). It is particularly preferred that (AA)_(x) ispresent as ligand or part of the repetitive component [S—P²—S]_(n).

In this context it is particularly preferred that the amino acidcomponent (AA)_(x) contains or is flanked (e.g. terminally) by at leastone —SH containing moiety, which allows introducing this component(AA)_(x) via a disulfide bond into the polymeric carrier according toformula (I) as defined herein. In this context, the amino acid component(AA)_(x) may also be read as a component —S-(AA)_(x)- or —S-(AA)_(x)-S—,wherein S represents a —SH containing moiety (or, of course, one sulfurof a disulfide bond), e.g. a cysteine residue. In the specific case thatthe —SH containing moiety represents a cysteine, the amino acidcomponent (AA)_(x) may also be read as -Cys-(AA)_(x)- or-Cys-(AA)_(x)-Cys- wherein Cys represents Cysteine and provides for thenecessary —SH-moiety for a disulfide bond. (Accordingly,-Cys-(AA)_(x)-Cys- may also be written as (S-Cys)-(AA)_(x)-(Cys-S)— and-Cys-(AA)_(x)- may also be written as (S-Cys)-(AA)_(x)-).) The —SHcontaining moiety may be also introduced into the amino acid component(AA)_(x) using any of modifications or reactions as shown above forcomponents P¹, P² or P³. In the specific case that the amino acidcomponent (AA)_(x) is linked to two components of the inventivepolymeric carrier according to formula (I) it is preferred that (AA)_(x)contains at least two —SH-moieties, e.g. at least two Cysteines,preferably at its terminal ends. This is particularly preferred if(AA)_(x) is part of the repetitive component [S—P²—S]_(n).

In an alternative the amino acid component (AA)_(x) is introduced intothe inventive polymeric carrier according to formula (I) as definedherein via any chemical possible addition reaction. Therefore the aminoacid component (AA)_(x) contains at least one further functional moiety,which allows attaching same to a further component as defined herein,e.g. component P¹ or P³, P², L, or a further amino acid component(AA)_(x), etc. Such functional moieties may be selected fromfunctionalities which allow the attachment of further components, e.g.functionalities as defined herein, e.g. by amide formation (e.g.carboxylic acids, sulphonic acids, amines, etc.), by Michael addition(e.g. maleinimide moieties, α,β unsatured carbonyls, etc.), by clickchemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g.alkenes or alkines), imine or hydrozone formation (aldehydes or ketons,hydrazins, hydroxylamins, amines), complexation reactions (avidin,biotin, protein G) or components which allow S_(n)-type substitutionreactions (e.g. halogenalkans, thiols, alcohols, amines, hydrazines,hydrazides, sulphonic acid esters, oxyphosphonium salts) or otherchemical moieties which can be utilized in the attachment of furthercomponents.

The amino acid component (AA)_(x) may also occur as a mixed repetitiveamino acid component [(AA)_(x)]_(z) wherein the number of amino acidcomponents (AA)_(x) is further defined by z. In this context, z is aninteger and may be selected from a range of about 1 to 30, preferablyfrom a range of about 1 to 15, more preferably 1 to 10 or 1 to 5 andeven more preferably selected from a number selected from 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be selected from a rangeformed by any two of the afore mentioned values. Such a mixed repetitiveamino acid component [(AA)_(x)]_(z) may be used to integrate several ofthe same or different amino acid components (AA)_(x) as defined hereinin the inventive polymeric carrier. Preferably, in the mixed repetitiveamino acid component [(AA)_(x)]_(z) the amino acid component (AA)_(x)may contain or may be flanked (e.g. terminally) by at least one —SHcontaining moiety, preferably at least two —SH containing moieties asalready defined above, which allows coupling the different amino acidcomponents (AA)_(x) using a disulfide bond via a condensationpolymerization. Likewise as above, the mixed repetitive amino acidcomponent [(AA)_(x)]_(z) may also be read as [S-(AA)_(x)-S]_(z), whereinS represents a —SH containing moiety, e.g. a cysteine residue. In thespecific case that the —SH containing moiety represents a cysteine, themixed repetitive amino acid component [(AA)_(x)]_(z) may also be read as[Cys-(AA)_(x)-Cys]_(z), wherein Cys represents Cysteine and provides forthe necessary —SH-moiety for a disulfide bond. The —SH containing moietymay be also introduced into the amino acid component (AA)_(x) using anyof modifications or reactions as shown above for components P¹, P² orP³.

The amino acid component (AA)_(x) or the mixed repetitive amino acidcomponent [(AA)_(x)]_(z) may be provided with at least one —SH-moiety,e.g. in a form represented by formula (AA)_(x)-SH. Then, the component(AA)_(x) according to formula (AA)_(x)-SH or the mixed repetitive aminoacid component [(AA)_(x)]_(z) according to formula [(AA)_(x)]_(z)-SH,may be bound to any of components L, P¹, P² and/or P³ or anothercomponent (AA)_(x) via a disulfide bond. If bound to component P¹ and/orcomponent P³, components P¹ and/or P³ preferably exhibit at least two—SH-moieties to allow further binding of components P¹ and/or P³ to acomponent P² via a —SH-moiety forming a disulfide bond (see above). Theamino acid component (AA)_(x) in a form represented by formula(AA)_(x)-SH or the mixed repetitive amino acid component [(AA)_(x)]_(z)according to formula [(AA)_(x)]_(z)—SH may be also used to terminate acondensation reaction due to its single —SH moiety. In this case, theamino acid component (AA)_(x) in a form represented by formula(AA)_(x)-SH is preferably coupled terminally to components P¹ and/or P³.The amino acid component (AA)_(x) in a form represented by formula(AA)_(x)-SH or the mixed repetitive amino acid component [(AA)_(x)]_(z)according to formula [(AA)_(x)]_(z)—SH may be also used to bindinternally to any of components L, P¹, P² and/or P³ or a furthercomponent (AA)_(x) via a further internal —SH-moiety of any ofcomponents L, P¹, P² and/or P³ or (AA)_(x).

Furthermore, the amino acid component (AA)_(x) may be provided with two—SH-moieties (or even more), e.g. in a form represented by formulaHS-(AA)_(x)-SH. Additionally, the mixed repetitive amino acid component[(AA)_(x)]_(z) may be provided with two —SH-moieties (or even more),e.g. in a form represented by formula HS-[(AA)_(x)]_(z)—SH, to allowbinding to two functionalities via disulfide bonds, e.g. if the aminoacid component (AA)_(x) or the mixed repetitive amino acid component[(AA)_(x)]_(z) is used as a linker between two further components (e.g.as a linker between components L and P¹, between components P¹ and P²,in or as a part of repetitive component [S—P²—S]_(n), between componentsP² and P³ and/or between components P³ and L). In this case, one —SHmoiety is preferably protected in a first step using a protecting groupas known in the art, leading to an amino acid component (AA)_(x) offormula HS-(AA)_(x)-S-protecting group or to a mixed repetitive aminoacid component [(AA)_(x)]_(z) of formula HS-[(AA)_(x)]_(z)-S-protectinggroup. Then, the amino acid component (AA)_(x) or the mixed repetitiveamino acid component MALL may be bound to a component L, P¹, P² and/orP³, to form a first disulfide bond via the non-protected —SH moiety. Theprotected-SH-moiety is then typically deprotected and bound to a furtherfree —SH-moiety of a further component L, P¹, P² and/or P³ to form asecond disulfide bond. In the case that the amino acid component(AA)_(x) or the mixed repetitive amino acid component MALL is part ofthe repetitive component [S—P²—S]_(n) it is preferred that the formationof the disulfide bonds between (AA)_(x) and P² concurrently occurs withthe polycondensation reaction of the repetitive component [S—P²—S]_(n)and therefore no protection of the at least two terminal —SH-moieties isnot necessary.

Alternatively, the amino acid component (AA)_(x) or the mixed repetitiveamino acid component [(AA)_(x)]_(z) may be provided with otherfunctionalities as already described above for components P¹ and P²and/or P³, which allow binding of the amino acid component (AA)_(x) orbinding of the mixed repetitive amino acid component [(AA)_(x)]_(z) toany of components P¹, P² and/or P³ or (AA)_(x) and optionally tocomponent L.

Thus, according to the present invention, the amino acid component(AA)_(x) and/or the mixed repetitive amino acid component [(AA)_(x)]_(z)may be bound to P¹, P², P³, (AA)_(x) and/or L with or without using adisulfide linkage. Binding without using a disulfide linkage may beaccomplished by any of the reactions described above, preferably bybinding the amino acid component (AA)_(x) or the mixed repetitive aminoacid component [(AA)_(x)]_(z) to P¹, P², P³, (AA)_(x) and/or L using anamid-chemistry as defined herein. If desired or necessary, the otherterminus of the amino acid component (AA)_(x) or the mixed repetitiveamino acid component [(AA)_(x)]_(z), e.g. the N- or C-terminus, may beused to couple another component, e.g. a ligand L. For this purpose, theother terminus of the amino acid component (AA)_(x) or the mixedrepetitive amino acid component [(AA)_(x)]_(z) preferably comprises oris modified to comprise a further functionality, e.g. an alkyn-species(see above), which may be used to add the other component via e.g.click-chemistry. Such a construct, e.g. L-(AA)_(x)-P¹—S— orL-[(AA)_(x)]_(z)-P¹—S—, may be used to terminate the polymerizationcondensation reaction of repetitive component [S—P²—S]_(n). If theligand is bound via an acid-labile bond, the bond may be cleaved off inthe endosome and the inventive polymeric carrier presents amino acidcomponent (AA)_(x) or the mixed repetitive amino acid component[(AA)_(x)]_(z) at its surface.

The amino acid component (AA)_(x) or the mixed repetitive amino acidcomponent [(AA)_(x)]_(z) may occur as a further component of genericformula (I) above, e.g. as a linker between components P¹ or P³ and P²,as a linker between components L and P¹ or P² or as an additionalcomponent of the repetitive component [S—P²—S]_(n).

According to a first alternative, such an amino acid component (AA)_(x)or the mixed repetitive amino acid component [(AA)_(x)]_(z) may bepresent as a linker between components P¹ or P³ and component P². Thisis preferably represented in the context of the entire inventivepolymeric carrier according to formula (I) by following formulae:

L-P¹—S—S-(AA)_(x)S—[S—P²—S]_(n)—S-(AA)_(x)-S—S—P³-L, or

L-P¹—S—[S-(AA)_(x)S]_(z)—[S—P²—S]_(n)—[S-(AA)_(x)-S]_(z)—S—P³-L,

wherein n, x, z, S, L, AA, P¹, P² and P³ are preferably as definedherein. In the above formulae, the term “—S—S—” represents a disulfidebond, wherein this at least one sulfur of the disulfide bond may also beprovided by a cysteine. In this case, the term “—S—S—” in these formulaemay also be written as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In thiscontext, the term “-Cys-Cys-” does not represent a peptide bond but alinkage of two cysteines via their —SH-moieties to form a disulfidebond. Accordingly, the term “-Cys-Cys-” may also be understood generallyas “-(Cys-S)—(S-Cys)-”, wherein in this specific case S indicates thesulfur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and“—Cys-S” indicate a disulfide bond between a —SH containing moiety and acysteine, which may also be written as “—S—(S—Cys)” and “-(Cys-S)—S”.

According to a second alternative, such an amino acid component (AA)_(x)or the mixed repetitive amino acid component [(AA)_(x)]_(z) may bepresent as a linker between components P¹ or P³ and component L. This ispreferably represented in the context of the entire inventive polymericcarrier according to formula (I) by following formulae:

L-(AA)_(x)-P¹—S—[S—P²—S]_(n)—S—P³-(AA)_(x)-L, or

L-[(AA)_(x)]_(z)-P¹—S—[S—P²—S]—S—P³-[(AA)_(x)]_(z)-L,

-   -   or alternatively

L-(AA)_(x)-S—S—P¹—S—[S—P²—S]_(n)—S—P³—S—S-(AA)_(x)-S—S-L, or

L-S—S-(AA)_(x)-S—S—P¹—S—[S—P²—S]_(n)—S—P³—S—S-(AA)_(x)-S—S-L, or

L-S[S-(AA)_(x)-S]_(z)—S—P¹—S—[S—P²—S]—S—P³—S—[S-(AA)_(x)-S]_(z)—S-L,etc.

wherein n, x, z, S, L, AA, P¹, P² and P³ are preferably as definedherein. In the above formulae, the term “—S—S—” represents a disulfidebond, as already defined above.

According to a third alternative, such an amino acid component (AA)_(x)or the mixed repetitive amino acid component [(AA)_(x)]_(z) may bepresent as a part of components P¹ and/or P³, wherein the amino acidcomponent (AA)_(x) may be directly bound to (e.g. the terminus of)component P¹ and/or P³ without a further ligand L. In this case the(AA)_(x) component may be in the form of a ligand as defined above. Thisis preferably represented in the context of the entire inventivepolymeric carrier according to formula (I) by following formulae:

(AA)_(x)-P¹—S—[S—P²—S]_(n)—S—P³-(AA)_(x), or

[(AA)_(x)]_(z)—P¹—S—[S—P²—S]_(n)—S—P³-[(AA)_(x)]_(z), or

or alternatively

(AA)_(x)-S—S—P¹—S[S—P²—S_(n)]—S—P³—S—S-(AA)_(x), or

H—[S-(AA)_(x)-S]_(z)—S—P¹—S—[S—P²—S]_(n)—S—S—P³—S—[S-(AA)_(x)-S]_(z)—H,

wherein n, x, z, S, AA, P¹, P² and P³ are preferably as defined herein.In the above formulae, the term “—S—S—” represents a disulfide bond, asalready defined above. The free —SH moiety at the terminal ends in thelast formula may also be terminated using a monothiol compound asdefined herein.

According to a fourth and particularly preferred alternative, the aminoacid component (AA)_(x), preferably written as S-(AA)_(x)-S or[S-(AA)_(x)-S] may be used to modify component P², particularly thecontent of component S—P²—S in repetitive component [S—P²—S]_(n) offormula (I) above. This may be represented in the context of the entirepolymeric carrier according to formula (I) e.g. by following formula(Ia):

L-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-L,

wherein x, S, L, AA, P¹, P² and P³ are preferably as defined herein. Informula (Ia) above, any of the single components [S—P²—S] and[S-(AA)_(x)-S] may occur in any order in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}. The numbers of single components[S—P²—S] and [S-(AA)_(x)-S] in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} are determined by integers a and b,wherein a+b=n. n is an integer and is defined as above for formula (I).

a is an integer, typically selected independent from integer b from arange of about 1 to 50, preferably from a range of about 1, 2 or 3 to30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or arange of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4,or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. arange of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range ofabout 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to11 or 7 to 10. Most preferably, a is in a range of about 1, 2, 3, 4, or5 to 10, more preferably in a range of about 1, 2, 3, or 4 to 9, in arange of about 1, 2, 3, or 4 to 8, or in a range of about 1, 2, or 3 to7.

b is an integer, typically selected independent from integer a from arange of about 0 to 50 or 1 to 50, preferably from a range of about 0,1, 2 or 3 to 30, more preferably from a range of about 0, 1, 2, 3, 4, or5 to 25, or a range of about 0, 1, 2, 3, 4, or 5 to 20, or a range ofabout 0, 1, 2, 3, 4, or 5 to 15, or a range of about 0, 1, 2, 3, 4, or 5to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or arange of about 6 to 11 or 7 to 10. Most preferably, b is in a range ofabout 1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2,3, or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range ofabout 1, 2, or 3 to 7.

In the above formula, the term “—S—S—” (the brackets are omitted forbetter readability) represents a disulfide bond as already definedabove.

The modification of component P², particularly of component S—P²—S ofrepetitive component [S—P²—S]_(n), by “diluting” same with amino acidcomponents (AA)_(x) may be also realized in the context of any of theafore mentioned alternatives of the entire polymeric carrier accordingto formula (I),

L-P¹—S—S-(AA)_(x)-S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S-(AA)_(x)-S—S—P³-L,or

L-P¹—S—[S-(AA)_(x)-S]_(z)—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—[S-(AA)_(x)-S]—S—P³-L,or

L-(AA)_(x)-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-(AA)_(x)-L, or

L-[(AA)_(x)]_(z)—P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-[(AA)_(x)]_(z)-L,or

L-(AA)_(x)-S—S—P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³—S—S-(AA)_(x)-S—S-L,or

L-S—S-(AA)_(x)-S—S—P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³—S—S-(AA)_(x)-S—S-L,or

L-S—[S-(AA)_(x)-S]_(z)—S—P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³—S—[S-(AA)_(x)-S]_(z)—S-L,or

(AA)_(x)-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-(AA)_(x), or

[(AA)_(x)]_(z)—P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-[(AA)_(x)]_(z),or

(AA)_(x)-S—S—P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³—S—S-(AA)_(x), or

H—[S-(AA)_(x)-S]_(z)—S—P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³—S—[S-(AA)_(x)-S]_(z)—H,

wherein n, x, z, a, b, S, L, AA, P¹, P² and P³ are preferably as definedherein. Likewise, the term “—S—S—” represents a disulfide bond and ispreferably as defined herein.

In the above alternatives, wherein the component [S—P²—S] is preferably“diluted” with amino acid components [S-(AA)_(x)-S], the ratio isdetermined by integers a and b, wherein a+b=n. Preferably, integers aand b are selected such that the cationic binding properties ofcomponent [S—P²—S] are not lost but remain to a minimum extent insubformula/component {[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}. This allows toweaken (“dilute”) the cationic binding strength of component [S—P²—S] inrepetitive component [S—P²—S]_(n) of inventive polymeric carrier offormula (I) to a desired extent.

In this specific context the (desired) cationic binding strength ofsubformula/component {([S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} may be determinedusing different methods.

According to a first alternative, component P² of formula (I) of thepresent invention is particularly preferable a cationic or polycationicpeptide as defined herein. Furthermore, the amino acid component(AA)_(x), preferably written as [S-(AA)_(x)-S], typically resembles apeptide sequence. In this specific case, the cationic properties ofsubformula/component {[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} may be determinedupon their content of cationic amino acids in the entiresubformula/component. Preferably, the content of cationic amino acids insubformula/component {[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} is at least 10%,20%, or 30%, preferably at least 40%, more preferably at least 50%, 60%or 70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%,98%, 99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about10% to 90%, more preferably in the range of about 15% to 75%, evenpreferably in the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, orin a range formed by any two of the afore mentioned values, provided,that the content of all amino acids, e.g. cationic, lipophilic,hydrophilic, aromatic and further amino acids, in the entiresubformula/component {[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} is 100%.

According to a second alternative, component P² of formula (I) of thepresent invention is particularly preferable a cationic or polycationicpolymer as defined herein. The amino acid component (AA)_(x), preferablywritten as [S-(AA)_(x)-S], typically resembles a peptide sequence. Inthis specific case, the cationic properties of subformula/component{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} may be determined upon their content ofcationic charges in the entire subformula/component. Preferably, thecontent of cationic charges in subformula/component{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} at a (physiological) pH as definedherein is at least 10%, 20%, or 30%, preferably at least 40%, morepreferably at least 50%, 60% or 70%, but also preferably at least 80%,90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, ormay be in the range of about 10% to 90%, more preferably in the range ofabout 15% to 75%, even preferably in the range of about 20% to 50%, e.g.20, 30, 40 or 50%, or in a range formed by any two of the aforementioned values, provided, that the content of all charges, e.g.positive and negative charges at a (physiological) pH as defined herein,in the entire subformula/component {[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} is100%.

In the context of the present invention, the amino acid component(AA)_(x) may be selected from the following alternatives.

According to a first alternative, the amino acid component (AA)_(x) maybe an aromatic amino acid component (AA)_(x). The incorporation ofaromatic amino acids or sequences as an amino aromatic acid component(AA)_(x) into the inventive polymeric carrier according to formula (I)of the present invention enables a different (second) binding of theinventive polymeric carrier to the nucleic acid due to interactions ofthe aromatic amino acids with the bases of the nucleic acid cargo incontrast to the binding thereof by cationic charged sequences of thepolymeric carrier molecule to the phosphate backbone. This interactionmay occur e.g. by intercalations or by minor or major groove binding.This kind of interaction is not prone to decompaction by anioniccomplexing partners (e.g. Heparin, Hyaluronic acids) which are foundmainly in the extracellular matrix in vivo and is also less susceptibleto salt effects.

For this purpose, the amino acid AA in the aromatic amino acid component(AA)_(x) may be selected from either the same or different aromaticamino acids e.g. selected from Trp, Tyr, or Phe. Alternatively, theamino acid AA (or the entire aromatic amino acid component (AA)_(x)) maybe selected from following peptide combinations Trp-Tyr, Tyr-Trp,Trp-Trp, Tyr-Tyr, Trp-Tyr-Trp, Tyr-Trp-Tyr, Trp-Trp-Trp, Tyr-Tyr-Tyr,Trp-Tyr-Trp-Tyr, Tyr-Trp-Tyr-Trp, Trp-Trp-Trp-Trp, Phe-Tyr, Tyr-Phe,Phe-Phe, Phe-Tyr-Phe, Tyr-Phe-Tyr, Phe-Phe-Phe, Phe-Tyr-Phe-Tyr,Tyr-Phe-Tyr-Phe, Phe-Phe-Phe-Phe, Phe-Trp, Trp-Phe, Phe-Phe,Phe-Trp-Phe, Trp-Phe-Trp, Phe-Trp-Phe-Trp, Trp-Phe-Trp-Phe, orTyr-Tyr-Tyr-Tyr, etc. (SEQ ID NOs: 85-112) or combinations thereof.

Additionally, the aromatic amino acid component (AA)_(x) may contain ormay be flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of generic formula (I)above, e.g. as a linker or more preferably as a component of therepetitive component [S—P²—S]_(n). Such a —SH containing moiety may beany moiety as defined herein suitable to couple one component as definedherein to a further component as defined herein. As an example, such a—SH containing moiety may be a cysteine. Then, e.g. the aromatic aminoacid component (AA)_(x) may be selected from e.g. peptide combinationsCys-Tyr-Cys, Cys-Trp-Cys, Cys-Trp-Tyr-Cys, Cys-Tyr-Trp-Cys,Cys-Trp-Trp-Cys, Cys-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Cys,Cys-Tyr-Trp-Tyr-Cys, Cys-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Cys,Cys-Trp-Tyr-Trp-Tyr-Cys, Cys-Tyr-Trp-Tyr-Trp-Cys,Cys-Trp-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Tyr-Cys, Cys-Phe-Cys,Cys-Phe-Tyr-Cys, Cys-Tyr-Phe-Cys, Cys-Phe-Phe-Cys, Cys-Tyr-Tyr-Cys,Cys-Phe-Tyr-Phe-Cys, Cys-Tyr-Phe-Tyr-Cys, Cys-Phe-Phe-Phe-Cys,Cys-Tyr-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Tyr-Cys, Cys-Tyr-Phe-Tyr-Phe-Cys,or Cys-Phe-Phe-Phe-Phe-Cys, Cys-Phe-Trp-Cys, Cys-Trp-Phe-Cys,Cys-Trp-Trp-Cys, Cys-Phe-Trp-Phe-Cys, Cys-Trp-Phe-Trp-Cys,Cys-Phe-Trp-Phe-Trp-Cys, Cys-Trp-Phe-Trp-Phe-Cys, etc. (SEQ ID NOs:113-145) or combinations thereof. Each Cys above may also be replaced byany modified peptide or chemical compound carrying a free —SH-moiety asdefined herein.

Additionally, the aromatic amino acid component (AA)_(x) may contain atleast one proline, which may serve as a structure breaker of longerrepetitive sequences of Trp, Tyr and Phe in the aromatic amino acidcomponent (AA)_(x), preferably two, three or more prolines.

According to a second alternative, the amino acid component (AA)_(x) maybe a hydrophilic (and preferably non charged polar) amino acid component(AA)_(x). The incorporation of hydrophilic (and preferably non chargedpolar) amino acids or sequences as amino hydrophilic (and preferably noncharged polar) acid component (AA)_(x) into the inventive polymericcarrier according to formula (I) of the present invention enables a moreflexible binding to the nucleic acid cargo. This leads to a moreeffective compaction of the nucleic acid cargo and hence to a betterprotection against nucleases and unwanted decompaction. It also allowsprovision of a (long) inventive polymeric carrier according to formula(I) which exhibits a reduced cationic charge over the entire carrier orpreferably within repetitive component [S—P²—S]_(n) and in this contextto better adjusted binding properties, if desired or necessary.

For this purpose, the amino acid AA in the hydrophilic (and preferablynon charged polar) amino acid component (AA)_(x) may be selected fromeither the same or different hydrophilic (and preferably non chargedpolar) amino acids e.g. selected from Thr, Ser, Asn or Gln.Alternatively, the amino acid AA (or the entire hydrophilic (andpreferably non charged polar) amino acid component (AA)_(x)) may beselected from following peptide combinations Ser-Thr, Thr-Ser, Ser-Ser,Thr-Thr, Ser-Thr-Ser, Thr-Ser-Thr, Ser-Ser-Ser, Thr-Thr-Thr,Ser-Thr-Ser-Thr, Thr-Ser-Thr-Ser, Ser-Ser-Ser-Ser, Thr-Thr-Thr-Thr,Gln-Asn, Asn-Gln, Gln-Gln, Asn-Asn, Gln-Asn-Gln, Asn-Gln-Asn,Gln-Gln-Gln, Asn-Asn-Asn, Gln-Asn-Gln-Asn, Asn-Gln-Asn-Gln,Gln-Gln-Gln-Gln, Asn-Asn-Asn-Asn, Ser-Asn, Asn-Ser, Ser-Ser, Asn-Asn,Ser-Asn-Ser, Asn-Ser-Asn, Ser-Ser-Ser, Asn-Asn-Asn, Ser-Asn-Ser-Asn,Asn-Ser-Asn-Ser, Ser-Ser-Ser-Ser, or Asn-Asn-Asn-Asn, etc. (SEQ ID NOs:146-181) or combinations thereof.

Additionally, the hydrophilic (and preferably non charged polar) aminoacid component (AA)_(x) may contain or may be flanked by a —SHcontaining moiety, which allows introducing this component via adisulfide bond as a further part of generic formula (I) above, e.g. as alinker or more preferably as component of the repetitive component[S—P²—S]_(n). Such a —SH containing moiety may be any moiety as definedherein suitable to couple one component as defined herein to a furthercomponent as defined herein. As an example, such a —SH containing moietymay be a cysteine. Then, e.g. the hydrophilic (and preferably noncharged polar) amino acid component (AA)_(x) may be selected from e.g.peptide combinations Cys-Thr-Cys, Cys-Ser-Cys, Cys-Ser-Thr-Cys,Cys-Thr-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Thr-Thr-Cys, Cys-Ser-Thr-Ser-Cys,Cys-Thr-Ser-Thr-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Cys,Cys-Ser-Thr-Ser-Thr-Cys, Cys-Thr-Ser-Thr-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Thr-Cys, Cys-Asn-Cys,Cys-Gln-Cys, Cys-Gln-Asn-Cys, Cys-Asn-Gln-Cys, Cys-Gln-Gln-Cys,Cys-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Cys, Cys-Asn-Gln-Asn-Cys,Cys-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Asn-Cys,Cys-Asn-Gln-Asn-Gln-Cys, Cys-Gln-Gln-Gln-Gln-Cys,Cys-Asn-Asn-Asn-Asn-Cys, Cys-Asn-Cys, Cys-Ser-Cys, Cys-Ser-Asn-Cys,Cys-Asn-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Cys,Cys-Asn-Ser-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Cys,Cys-Ser-Asn-Ser-Asn-Cys, Cys-Asn-Ser-Asn-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, or Cys-Asn-Asn-Asn-Asn-Cys, etc. (SEQ ID NOs:182-223) or combinations thereof. Each Cys above may also be replaced byany modified peptide or chemical compound carrying a free —SH-moiety asdefined herein.

Additionally, the hydrophilic (and preferably non charged polar) aminoacid component (AA)_(x) may contain at least one proline, which mayserve as a structure breaker of longer repetitive sequences of Ser, Thrand Asn in the hydrophilic (and preferably non charged polar) amino acidcomponent (AA)_(x), preferably two, three or more prolines.

According to a third alternative, the amino acid component (AA)_(x) maybe a lipophilic amino acid component (AA)_(x). The incorporation oflipophilic amino acids or sequences as amino lipophilic acid component(AA))_(x) into the inventive polymeric carrier according to formula (I)of the present invention enables a stronger compaction of the nucleicacid cargo and/or the polymeric carrier according to formula (I) and itsnucleic acid cargo when forming a complex. This is particularly due tointeractions of one or more polymer strands of the inventive polymericcarrier, particularly of lipophilic sections of lipophilic amino acidcomponent (AA)_(x), preferably in the context of subformula/component{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}, and the nucleic acid cargo. Thisinteraction will preferably add an additional stability to the complexbetween the polymeric carrier according to formula (I) and its nucleicacid cargo. This stabilization may somehow be compared to a sort of noncovalent crosslinking between different polymerstrands. Especially inaqueous environment this interaction is typically strong and provides asignificant effect.

For this purpose, the amino acid AA in the lipophilic amino acidcomponent (AA)_(x) may be selected from either the same or differentlipophilic amino acids e.g. selected from Leu, Val, Ile, Ala, Met.Alternatively, the amino acid AA (or the entire lipophilic amino acidcomponent (AA)_(x)) may be selected from following peptide combinationsLeu-Val, Val-Leu, Leu-Leu, Val-Val, Leu-Val-Leu, Val-Leu-Val,Leu-Leu-Leu, Val-Val-Val, Leu-Val-Leu-Val, Val-Leu-Val-Leu,Leu-Leu-Leu-Leu, Val-Val-Val-Val, Ile-Ala, Ala-Ile, Ile-Ile, Ala-Ala,Ile-Ala-Ile, Ala-Ile-Ala, Ile-Ile-Ile, Ala-Ala-Ala, Ile-Ala-Ile-Ala,Ala-Ile-Ala-Ile, Ile-Ile-Ile-Ile, Ala-Ala-Ala-Ala, Met-Ala, Ala-Met,Met-Met, Ala-Ala, Met-Ala-Met, Ala-Met-Ala, Met-Met-Met, Ala-Ala-Ala,Met-Ala-Met-Ala, Ala-Met-Ala-Met, or Met-Met-Met-Met etc. (SEQ ID NOs:224-258) or combinations thereof.

Additionally, the lipophilic amino acid component (AA)_(x) may containor may be flanked by a —SH containing moiety, which allows introducingthis component via a disulfide bond as a further part of generic formula(I) above, e.g. as a linker or more preferably as component of therepetitive component [S—P²—S]_(n). Such a —SH containing moiety may beany moiety as defined herein suitable to couple one component as definedherein to a further component as defined herein. As an example, such a—SH containing moiety may be a cysteine. Then, e.g. the lipophilic aminoacid component (AA)_(x) may be selected from e.g. peptide combinationsCys-Val-Cys, Cys-Leu-Cys, Cys-Leu-Val-Cys, Cys-Val-Leu-Cys,Cys-Leu-Leu-Cys, Cys-Val-Val-Cys, Cys-Leu-Val-Leu-Cys,Cys-Val-Leu-Val-Cys, Cys-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Cys,Cys-Leu-Val-Leu-Val-Cys, Cys-Val-Leu-Val-Leu-Cys,Cys-Leu-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Val-Cys, Cys-Ala-Cys,Cys-Ile-Cys, Cys-Ile-Ala-Cys, Cys-Ala-Ile-Cys, Cys-Ile-Ile-Cys,Cys-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Cys, Cys-Ala-Ile-Ala-Cys,Cys-Ile-Ile-Ile-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Ala-Cys,Cys-Ala-Ile-Ala-Ile-Cys, Cys-Ile-Ile-Ile-Ile-Cys, orCys-Ala-Ala-Ala-Ala-Cys, Cys-Met-Cys, Cys-Met-Ala-Cys, Cys-Ala-Met-Cys,Cys-Met-Met-Cys, Cys-Ala-Ala-Cys, Cys-Met-Ala-Met-Cys,Cys-Ala-Met-Ala-Cys, Cys-Met-Met-Met-Cys, Cys-Ala-Ala-Ala-Cys,Cys-Met-Ala-Met-Ala-Cys, Cys-Ala-Met-Ala-Met-Cys,Cys-Met-Met-Met-Met-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, etc. (SEQ ID NOs:289-299) or combinations thereof. Each Cys above may also be replaced byany modified peptide or chemical compound carrying a free —SH-moiety asdefined herein.

Additionally, the lipophilic amino acid component (AA)_(x) may containat least one proline, which may serve as a structure breaker of longerrepetitive sequences of Leu, Val, Ile, Ala and Met in the lipophilicamino acid component (AA)_(x), preferably two, three or more prolines.

According to a fourth alternative, the amino acid component (AA)_(x) maybe a weak basic amino acid component (AA)_(x). The incorporation of weakbasic amino acids or sequences as weak basic amino acid component(AA)_(x) into the inventive polymeric carrier according to formula (I)of the present invention may serve as a proton sponge and facilitatesendosomal escape (also called endosomal release) (proton sponge effect).Incorporation of such a weak basic amino acid component (AA)_(x)preferably enhances transfection efficiency.

For this purpose, the amino acid AA in the weak basic amino acidcomponent (AA)_(x) may be selected from either the same or differentweak amino acids e.g. selected from histidine or aspartate (asparticacid). Alternatively, the weak basic amino acid AA (or the entire weakbasic amino acid component (AA)_(x)) may be selected from followingpeptide combinations Asp-His, His-Asp, Asp-Asp, His-His, Asp-His-Asp,His-Asp-His, Asp-Asp-Asp, His-His-His, Asp-His-Asp-His, His-Asp-His-Asp,Asp-Asp-Asp-Asp, or His-His-His-His, etc. (SEQ ID NOs: 300-311) orcombinations thereof.

Additionally, the weak basic amino acid component (AA)_(x) may containor may be flanked by a —SH containing moiety, which allows introducingthis component via a disulfide bond as a further part of generic formula(I) above, e.g. as a linker or more preferably as component of therepetitive component [S—P²—S]_(n). Such a —SH containing moiety may beany moiety as defined herein suitable to couple one component as definedherein to a further component as defined herein. As an example, such a—SH containing moiety may be a cysteine. Then, e.g. the weak basic aminoacid component (AA)_(x), may be selected from e.g. peptide combinationsCys-His-Cys, Cys-Asp-Cys, Cys-Asp-His-Cys, Cys-His-Asp-Cys,Cys-Asp-Asp-Cys, Cys-His-His-Cys, Cys-Asp-His-Asp-Cys,Cys-His-Asp-His-Cys, Cys-Asp-Asp-Asp-Cys, Cys-His-His-His-Cys,Cys-Asp-His-Asp-His-Cys, Cys-His-Asp-His-Asp-Cys,Cys-Asp-Asp-Asp-Asp-Cys, or Cys-His-His-His-His-Cys, etc. (SEQ ID NOs:312-325) or combinations thereof. Each Cys above may also be replaced byany modified peptide or chemical compound carrying a free —SH-moiety asdefined herein.

Additionally, the weak basic amino acid component (AA)_(x) may containat least one proline, which may serve as a structure breaker of longerrepetitive sequences of histidine or aspartate (aspartic acid) in theweak basic amino acid component (AA)_(x), preferably two, three or moreprolines.

Additionally, the inventive polymeric carrier according to formula (I)above (or according to any of its subformulas herein), may comprise asan additional component, preferably as a ligand L or as an amino acidcomponent (AA)_(x) a signal peptide, a localization signal or sequenceor a nuclear localization signal or sequence (NLS), which allows atranslocalization of the inventive polymeric carrier according toformula (I) above to a specific target, e.g. into the cell, into thenucleus, into the endosomal compartiment, sequences for themitochondrial matrix, localisation sequences for the plasma membrane,localisation sequences for the Golgi apparatus, the nucleus, thecytoplasm and the cytosceleton, etc. Such a signal peptide, localizationsignal or sequence or nuclear localization signal may be used for thetransport of any of the herein defined nucleic acids, preferably an RNAor a DNA, more preferably an shRNA or a pDNA, e.g. into the nucleus.Without being limited thereto, such a signal peptide, localizationsignal or sequence or nuclear localization signal may comprise, e.g.,localisation sequences for the endoplasmic reticulum. Particularlocalization signals or sequences or nuclear localization signals mayinclude e.g. KDEL (SEQ ID NO: 326), DDEL (SEQ ID NO: 327), DEEL (SEQ IDNO: 328), QEDL (SEQ ID NO: 329), RDEL (SEQ ID NO: 330), and GQNLSTSN(SEQ ID NO: 331), nuclear localisation sequences, including PKKKRKV (SEQID NO: 332), PQKKIKS (SEQ ID NO: 333), QPKKP (SEQ ID NO: 334), RKKR (SEQID NO: 335), RKKRRQRRRAHQ (SEQ ID NO: 336), RQARRNRRRRWRERQR (SEQ ID NO:337), MPLTRRRPAASQALAPPTP (SEQ ID NO: 338), GAALTILV (SEQ ID NO: 339),and GAALTLLG (SEQ ID NO: 340), localisation sequences for the endosomalcompartiment, including MDDQRDLISNNEQLP (SEQ ID NO: 341), localisationsequences for the mitochondrial matrix, includingMLFNLRXXLNNAAFRHGHNFMVRNFRCGQPLX (SEQ ID NO: 342), localisationsequences for the plasma membrane: GCVCSSNP (SEQ ID NO: 343), GQTVTTPL(SEQ ID NO: 344), GQELSQHE (SEQ ID NO: 345), GNSPSYNP (SEQ ID NO: 346),GVSGSKGQ (SEQ ID NO: 347), GQTITTPL (SEQ ID NO: 348), GQTLTTPL (SEQ IDNO: 349), GQIFSRSA (SEQ ID NO: 350), GQIHGLSP (SEQ ID NO: 351), GARASVLS(SEQ ID NO: 352), and GCTLSAEE (SEQ ID NO: 353), localisation sequencesfor the endoplasmic reticulum and the nucleus, including GAQVSSQK (SEQID NO: 354), and GAQLSRNT (SEQ ID NO: 355), localisation sequences forthe Golgi apparatus, the nucleus, the cytoplasm and the cytosceleton,including GNAAAAKK (SEQ ID NO: 356), localisation sequences for thecytoplasm and cytosceleton, including GNEASYPL (SEQ ID NO: 357),localisation sequences for the plasma membrane and cytosceleton,including GSSKSKPK (SEQ ID NO: 358), etc. Examples of secretory signalpeptide sequences as defined herein include, without being limitedthereto, signal sequences of classical or non-classical MHC-molecules(e.g. signal sequences of MHC I and II molecules, e.g. of the MHC classI molecule HLA-A*0201), signal sequences of cytokines orimmunoglobulines as defined herein, signal sequences of the invariantchain of immunoglobulines or antibodies as defined herein, signalsequences of Lamp1, Tapasin, Erp57, Calretikulin, Calnexin, and furthermembrane associated proteins or of proteins associated with theendoplasmic reticulum (ER) or the endosomal-lysosomal compartiment.Particularly preferably, signal sequences of MHC class I moleculeHLA-A*0201 may be used according to the present invention. Mostpreferably such an additional component may occur as component L asdefined herein. Alternatively, such an additional component may also bebound e.g. to a component L, P¹, P², r P³ or (AA)_(x) as defined herein,e.g. to a side chain of any of components L, P¹, P², P³ or (AA)_(x),preferably via a side chain of component P², or optionally as a linkerbetween components L and P¹ or P³ and L. The binding to any ofcomponents L, P¹, P², or P³ may also be accomplished using anacid-labile bond, preferably via a side chain of any of components L,P¹, P², P³, which allows to detach or release the additional componentat lower pH-values, e.g. at physiological pH-values as defined herein.

Additionally, the inventive polymeric carrier according to formula (I)above (or according to any of its subformulas herein), may comprisefurther functional peptides or proteins preferably as ligand or aminoacid component (AA)_(x), which may modulate the functionality of theinventive polymeric carrier accordingly. According to one alternative,such further functional peptides or proteins may comprise so called cellpenetrating peptides (CPPs) or cationic peptides for transportation.Particularly preferred are CPPs, which induce a pH-mediatedconformational change in the endosome and lead to an improved release ofthe inventive polymeric carrier (in complex with a nucleic acid) fromthe endosome by insertion into the lipid layer of the liposome. Suchcalled cell penetrating peptides (CPPs) or cationic peptides fortransportation, may include, without being limited thereto protamine,nucleoline, spermine or spermidine, poly-L-lysine (PLL), basicpolypeptides, poly-arginine, cell penetrating peptides (CPPs), chimericCPPs, such as Transportan, or MPG peptides, HIV-binding peptides, Tat,HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of thepenetratin family, e.g. Penetratin, Antennapedia-derived peptides(particularly from Drosophila antennapecha), pAntp, plsl, etc.,antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1),pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Proline-richpeptides, Loligomers, Arginine-rich peptides, Calcitonin-peptides, FGF,Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived oranalog peptides, HSV, VP22 (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs, PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitoninpeptide(s), etc. Likewise, such an additional component may occur ascomponent L or (AA)_(x), as defined herein. Alternatively, such anadditional component may also be bound to a component L, P¹, P², P³ or(AA)_(x), as defined herein, e.g. to a side chain of any of componentsL, P¹, P², P³, or (AA)_(x), preferably via a side chain of component P²,or optionally as a linker between components L and P¹ or P³ and L. Thebinding to any of components L, P¹, P², P³ or (AA)_(x) may also beaccomplished using an acid-labile bond, preferably via a side chain ofany of components L, P¹, P², P³, or (AA)_(x) which allows to detach orrelease the additional component at lower pH-values, e.g. atphysiological pH-values as defined herein. In this context it isparticularly preferred that this additional component occurs as ligand Lor as amino acid component (AA)_(x) of the repetitive component[S—P²—S]_(n) of formula (I).

According to a last alternative, the inventive polymeric carrieraccording to formula (I) above (or according to any of its subformulasherein), may comprise as an additional component, preferably as aminoacid component (AA)_(x), any peptide or protein which can execute anyfavorable function in the cell. Particularly preferred are peptides orproteins selected from therapeutically active proteins or peptides, fromantigens, e.g. tumour antigens, pathogenic antigens (animal antigens,viral antigens, protozoal antigens, bacterial antigens, allergicantigens), autoimmune antigens, or further antigens, from allergens,from antibodies, from immunostimulatory proteins or peptides, fromantigen-specific T-cell receptors, or from any other protein or peptidesuitable for a specific (therapeutic) application as defined below forcoding nucleic acids. Particularly preferred are peptide epitopes fromantigens as defined herein. Likewise, such an additional component mayoccur preferably as (AA)_(x) as defined herein. Alternatively, such anadditional component may also be bound to a component L, P¹, P², P³ or(AA)_(x) as defined herein, e.g. to a side chain of any of components L,P¹, P², P³, or (AA)_(x), preferably via a side chain of component P², oroptionally as a linker between components L and P¹ or P³ and L. Thebinding to any of components L, P¹, P², P³ or (AA)_(x) may also beaccomplished using an acid-labile bond, preferably via a side chain ofany of components L, P¹, P², P³, or (AA)_(x) which allows to detach orrelease the additional component at lower pH-values, e.g. atphysiological pH-values as defined herein. In this context it isparticularly preferred that this additional component occurs as aminoacid component (AA)_(x) of the repetitive component [S—P²—S]_(n) offormula (I).

The inventive polymeric carrier according to formula (I) may comprise atleast one of the above mentioned cationic or polycationic peptides,proteins or polymers or further components, e.g. (AA), wherein any ofthe above alternatives may be combined with each other, and may beformed by polymerizing same in a polymerization condensation reactionvia their —SH-moieties.

The object underlying the present invention is furthermore solvedaccording to a second embodiment of the present invention by theinventive polymeric carrier cargo complex formed by the nucleic acidcargo and a polymeric carrier molecule according to generic formula (I)L-P¹—S—[S—P²—S]_(n)—S—P³-L as defined herein (or according to any of itssubformulas as defined herein). This complex may also be termed“complexed nucleic acid” for the purposes of the present application.

In the inventive polymeric carrier cargo complex, the polymeric carriermolecule according to generic formula (I) L-P¹—S—[S—P²—S]_(n)—S—P³-L asdefined herein (or according to any of its subformulas herein) and thenucleic acid cargo are typically provided in a molar ratio of about 5 to10000, preferably in a molar ratio of about 5 to 5000, more preferablyin a molar ratio of about 5 to 2500, even more preferably in a molarratio of about 5 to 2000, and most preferably in a molar ratio of about5 to 1000 of inventive polymeric carrier molecule:nucleic acid, or in amolar ratio of about 50 to 1000 of inventive polymeric carriermolecule:nucleic acid, e.g. in a molar ratio of about 10 to 5000, in amolar ratio of about 20 to 2500, in a molar ratio of about 25 to 2000 ofinventive polymeric carrier molecule:nucleic acid.

Furthermore, in the inventive polymeric carrier cargo complex, thepolymeric carrier molecule according to generic formula (I)L-P¹—S—[S—P²—S]_(n)—S—P³-L as defined herein (or according to any of itssubformulas herein) and the nucleic acid cargo are preferably providedin an N/P-ratio of about 0.1 to 20, preferably in an N/P-ratio of about0.2 to 12, and even more preferably in an N/P-ratio of about 0.4 to 10or 0.6 to 5. In this context, an N/P-ratio is defined as thenitrogen/phosphate ratio (N/P-ratio) of the entire inventive polymericcarrier cargo complex. This is typically illustrative for thecontent/amount of peptides, if peptides are used, in the inventivepolymeric carrier and characteristic for the content/amount of nucleicacids bound or complexed in the inventive polymeric carrier cargocomplex. It may be calculated on the basis that, for example, 1 μg RNAtypically contains about 3 nmol phosphate residues, provided that theRNA exhibits a statistical distribution of bases. Additionally, 1 μgpeptide typically contains about x*1 μg/M (peptide) nmol nitrogenresidues, dependent on the molecular weight and the number x of its(cationic) amino acids.

In the context of the present invention such a nucleic acid cargo of theinventive polymeric carrier cargo complex formed by the nucleic acidcargo and a polymeric carrier molecule according to generic formula (I)(or according to any of its subformulas herein) may be any suitablenucleic acid, selected e.g. from any DNA, preferably, without beinglimited thereto, e.g. genomic DNA, single-stranded DNA molecules,double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, apDNA, immunostimulating DNA or may be selected e.g. from any PNA(peptide nucleic acid) or may be selected e.g. from any RNA, preferably,without being limited thereto, a coding RNA, a messenger RNA (mRNA), ansiRNA, an shRNA, an antisense RNA, or riboswitches, immunostimulatingRNA (isRNA) ribozymes or aptamers; etc. The nucleic acid may also be aribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), ora viral RNA (vRNA). Preferably, the nucleic acid is RNA, more preferablya coding RNA. Even more preferably, the nucleic acid may be a (linear)single-stranded RNA, even more preferably an mRNA. In the context of thepresent invention, an mRNA is typically an RNA, which is composed ofseveral structural elements, e.g. an optional 5′-UTR region, an upstreampositioned ribosomal binding site followed by a coding region, anoptional 3′-UTR region, which may be followed by a poly-A tail (and/or apoly-C-tail). An mRNA may occur as a mono-, di-, or even multicistronicRNA, i.e. an RNA which carries the coding sequences of one, two or more(identical or different) proteins or peptides as defined herein. Suchcoding sequences in di-, or even multicistronic mRNA may be separated byat least one IRES (internal ribosomal entry site) sequence.

Furthermore, the nucleic acid of the inventive polymeric carrier cargocomplex formed by the nucleic acid cargo and a polymeric carriermolecule according to generic formula (I) (or according to any of itssubformulas herein) may be a single- or a double-stranded nucleic acid(molecule) (which may also be regarded as a nucleic acid (molecule) dueto non-covalent association of two single-stranded nucleic acid(s)(molecules)) or a partially double-stranded or partially single strandednucleic acid, which are at least partially self complementary (both ofthese partially double-stranded or partially single stranded nucleicacid molecules are typically formed by a longer and a shortersingle-stranded nucleic acid molecule or by two single stranded nucleicacid molecules, which are about equal in length, wherein onesingle-stranded nucleic acid molecule is in part complementary to theother single-stranded nucleic acid molecule and both thus form adouble-stranded nucleic acid molecule in this region, i.e. a partiallydouble-stranded or partially single stranded nucleic acid (molecule).Preferably, the nucleic acid (molecule) may be a single-stranded nucleicacid molecule. Furthermore, the nucleic acid (molecule) may be acircular or linear nucleic acid molecule, preferably a linear nucleicacid molecule.

Coding Nucleic Acids:

The nucleic acid molecule of the inventive polymeric carrier cargocomplex may encode a protein or a peptide, which may be selected,without being restricted thereto, e.g. from therapeutically activeproteins or peptides, selected e.g. from adjuvant proteins, fromantigens, e.g. tumour antigens, pathogenic antigens (e.g. selected, fromanimal antigens, from viral antigens, from protozoal antigens, frombacterial antigens), allergenic antigens, autoimmune antigens, orfurther antigens, from allergens, from antibodies, fromimmunostimulatory proteins or peptides, from antigen-specific T-cellreceptors, or from any other protein or peptide suitable for a specific(therapeutic) application, wherein the coding nucleic acid may betransported into a cell, a tissue or an organism and the protein may beexpressed subsequently in this cell, tissue or organism.

The coding region of the nucleic acid molecule of the inventivepolymeric carrier cargo complex may occur as a mono-, di-, or evenmulticistronic nucleic acid, i.e. a nucleic acid which carries thecoding sequences of one, two or more proteins or peptides. Such codingsequences in di-, or even multicistronic nucleic acids may be separatedby at least one internal ribosome entry site (IRES) sequence, or bysignal peptides which induce the cleavage of the resulting polypeptidewhich comprises several proteins or peptides.

In particular preferred aspects the encoded peptides or proteins areselected from human, viral, bacterial, protozoan proteins or peptides.

a) Therapeutically Active Proteins

In the context of the present invention, therapeutically active proteinsor peptides may be encoded by the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex. Therapeuticallyactive proteins are defined herein as proteins which have an effect onhealing, prevent prophylactically or treat therapeutically a disease,preferably as defined herein, or are proteins of which an individual isin need of. These may be selected from any naturally or syntheticallydesigned occurring recombinant or isolated protein known to a skilledperson from the prior art. Without being restricted theretotherapeutically active proteins may comprise proteins, capable ofstimulating or inhibiting the signal transduction in the cell, e.g.cytokines, lymphokines, monokines, growth factors, receptors, signaltransduction molecules, transcription factors, etc; anticoagulants;antithrombins; antiallergic proteins; apoptotic factors or apoptosisrelated proteins, therapeutic active enzymes and any protein connectedwith any acquired disease or any hereditary disease.

A therapeutically active protein, which may be encoded by the nucleicacid molecule of the herein defined inventive polymeric carrier cargocomplex, may also be an adjuvant protein. In this context, an adjuvantprotein is preferably to be understood as any protein, which is capableto elicit an innate immune response as defined herein. Preferably, suchan innate immune response comprises activation of a pattern recognitionreceptor, such as e.g. a receptor selected from the Toll-like receptor(TLR) family, including e.g. a Toll like receptor selected from humanTLR1 to TLR10 or from murine Toll like receptors TLR1 to TLR13. Morepreferably, the adjuvant protein is selected from human adjuvantproteins or from pathogenic adjuvant proteins, selected from the groupconsisting of, without being limited thereto, bacterial proteins,protozoan proteins, viral proteins, or fungal proteins, animal proteins,in particular from bacterial adjuvant proteins. In addition, nucleicacids encoding human proteins involved in adjuvant effects (e.g. ligandsof pattern recognition receptors, pattern recoginition receptors,proteins of the signal transduction pathways, transcription factors orcytokines) may be used as well.

b) Antigens

The nucleic acid molecule of the herein defined inventive polymericcarrier cargo complex may alternatively encode an antigen. According tothe present invention, the term “antigen” refers to a substance which isrecognized by the immune system and is capable of triggering anantigen-specific immune response, e.g. by formation of antibodies orantigen-specific T-cells as part of an adaptive immune response. In thiscontext an antigenic epitope, fragment or peptide of a protein meansparticularly B cell and T cell epitopes which may be recognized by Bcells, antibodies or T cells respectively.

In the context of the present invention, antigens as encoded by thenucleic acid molecule of the herein defined inventive polymeric carriercargo complex typically comprise any antigen, antigenic epitope orantigenic peptide, falling under the above definition, more preferablyprotein and peptide antigens, e.g. tumour antigens, allergenic antigens,auto-immune self-antigens, pathogenic antigens, etc. In particularantigens as encoded by the nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex may be antigens generatedoutside the cell, more typically antigens not derived from the hostorganism (e.g. a human) itself (i.e. non-self antigens) but ratherderived from host cells outside the host organism, e.g. viral antigens,bacterial antigens, fungal antigens, protozoological antigens, animalantigens, allergenic antigens, etc. Allergenic antigens (allergyantigens) are typically antigens, which cause an allergy in a human andmay be derived from either a human or other sources. Additionally,antigens as encoded by the nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex may be furthermore antigensgenerated inside the cell, the tissue or the body. Such antigens includeantigens derived from the host organism (e.g. a human) itself, e.g.tumour antigens, self-antigens or auto-antigens, such as auto-immuneself-antigens, etc., but also (non-self) antigens as defined herein,which have been originally been derived from host cells outside the hostorganism, but which are fragmented or degraded inside the body, tissueor cell, e.g. by (protease) degradation, metabolism, etc.

One class of antigens as encoded by the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex comprisestumour antigens. “Tumour antigens” are preferably located on the surfaceof the (tumour) cell. Tumour antigens may also be selected fromproteins, which are overexpressed in tumour cells compared to a normalcell. Furthermore, tumour antigens also include antigens expressed incells which are (were) not themselves (or originally not themselves)degenerated but are associated with the supposed tumour. Antigens whichare connected with tumour-supplying vessels or (re)formation thereof, inparticular those antigens which are associated with neovascularization,e.g. growth factors, such as VEGF, bFGF etc., are also included herein.Antigens connected with a tumour furthermore include antigens from cellsor tissues, typically embedding the tumour. Further, some substances(usually proteins or peptides) are expressed in patients suffering(knowingly or not-knowingly) from a cancer disease and they occur inincreased concentrations in the body fluids of said patients. Thesesubstances are also referred to as “tumour antigens”, however they arenot antigens in the stringent meaning of an immune response inducingsubstance. The class of tumour antigens can be divided further intotumour-specific antigens (TSAs) and tumour-associated-antigens (TAAs).TSAs can only be presented by tumour cells and never by normal “healthy”cells. They typically result from a tumour specific mutation. TAAs,which are more common, are usually presented by both tumour and healthycells. These antigens are recognized and the antigen-presenting cell canbe destroyed by cytotoxic T cells. Additionally, tumour antigens canalso occur on the surface of the tumour in the form of, e.g., a mutatedreceptor. In this case, they can be recognized by antibodies.

According to a preferred aspect, such tumor antigens as encoded by thenucleic acid of the inventive polymeric carrier cargo complex areselected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIPHPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m,B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m,CA 15-3/CA 27-29, CA 19-9, CA72-4, CAl25, calreticulin, CAMEL, CASP-8/m,cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4,CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28,CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6,Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN,EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1,EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gpl 00, GPC3, GPNMB/m, HAGE,HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R171, HLA-A11/m,HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6,HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5,immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205,KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1,MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16,MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1,MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A,MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, MEl/m, mesothelin,MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m,MUM-2/m, MUM-3/m, myosin class l/m, NA88-A,N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP,NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT,OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl,p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, Pim-1-Kinase,Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA,PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1,RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Spl 7, SSX-1,SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-2B,SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta,TGFbetaRll, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2,TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, and WT1, or a fragment,variant or epitope thereof. Epitopes typically comprise 5 to 15,preferably 5 to 12, more preferably 6 to 9 amino acids of the antigen,preferably in its native form.

According to another alternative, one further class of antigens asencoded by the nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex comprises allergenic antigens. Suchallergenic antigens may be selected from antigens derived from differentsources, e.g. from animals, plants, fungi, bacteria, etc. Allergens inthis context include e.g. grasses, pollens, molds, drugs, or numerousenvironmental triggers, etc. Allergenic antigens typically belong todifferent classes of compounds, such as nucleic acids and theirfragments, proteins or peptides and their fragments, carbohydrates,polysaccharides, sugars, lipids, phospholipids, etc. Of particularinterest in the context of the present invention are antigens, which maybe encoded by the nucleic acid molecule of the inventive polymericcarrier cargo complex, i.e. protein or peptide antigens and theirfragments or epitopes, or nucleic acids and their fragments,particularly nucleic acids and their fragments, encoding such protein orpeptide antigens and their fragments or epitopes.

c) Antibodies

According to a further alternative, the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may encode anantibody or an antibody fragment. According to the present invention,such an antibody may be selected from any antibody, e.g. anyrecombinantly produced or naturally occurring antibodies, known in theart, in particular antibodies suitable for therapeutic, diagnostic orscientific purposes, or antibodies which have been identified inrelation to specific cancer diseases. Herein, the term “antibody” isused in its broadest sense and specifically covers monoclonal andpolyclonal antibodies (including agonist, antagonist, and blocking orneutralizing antibodies) and antibody species with polyepitopicspecificity. According to the invention, the term “antibody” typicallycomprises any antibody known in the art (e.g. IgM, IgD, IgG, IgA and IgEantibodies), such as naturally occurring antibodies, antibodiesgenerated by immunization in a host organism, antibodies which wereisolated and identified from naturally occurring antibodies orantibodies generated by immunization in a host organism andrecombinantly produced by biomolecular methods known in the art, as wellas chimeric antibodies, human antibodies, humanized antibodies,bispecific antibodies, intrabodies, i.e. antibodies expressed in cellsand optionally localized in specific cell compartments, and fragmentsand variants of the aforementioned antibodies. In general, an antibodyconsists of a light chain and a heavy chain both having variable andconstant domains. The light chain consists of an N-terminal variabledomain, V_(L), and a C-terminal constant domain, C_(L). In contrast, theheavy chain of the IgG antibody, for example, is comprised of anN-terminal variable domain, V_(H), and three constant domains, C_(H)1,C_(H)2 and C_(H)3.

In the context of the present invention, antibodies as encoded by thenucleic acid molecule of the herein defined inventive polymeric carriercargo complex may preferably comprise full-length antibodies, i.e.antibodies composed of the full heavy and full light chains, asdescribed above. However, derivatives of antibodies such as antibodyfragments, variants or adducts may also be encoded by the nucleic acidmolecule of the herein defined inventive polymeric carrier cargocomplex. Antibody fragments are preferably selected from Fab, Fab′,F(ab′)₂, Fc, Facb, pFc′, Fd and Fv fragments of the aforementioned(full-length) antibodies. In general, antibody fragments are known inthe art. For example, a Fab (“fragment, antigen binding”) fragment iscomposed of one constant and one variable domain of each of the heavyand the light chain. The two variable domains bind the epitope onspecific antigens. The two chains are connected via a disulfide linkage.A scFv (“single chain variable fragment”) fragment, for example,typically consists of the variable domains of the light and heavychains. The domains are linked by an artificial linkage, in general apolypeptide linkage such as a peptide composed of 15-25 glycine, prolineand/or serine residues.

In the present context it is preferable that the different chains of theantibody or antibody fragment are encoded by a multicistronic nucleicacid molecule. Alternatively, the different strains of the antibody orantibody fragment are encoded by several monocistronic nucleic acid(s)(sequences).

siRNA:

According to a further alternative, the nucleic acid of the inventivepolymeric carrier cargo complex formed by the nucleic acid cargo and apolymeric carrier molecule according to generic formula (I) (oraccording to any of its subformulas herein) may be in the form of dsRNA,preferably siRNA. A dsRNA, or a siRNA, is of interest particularly inconnection with the phenomenon of RNA interference. The in vitrotechnique of RNA interference (RNAi) is based on double-stranded RNAmolecules (dsRNA), which trigger the sequence-specific suppression ofgene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp(2001) Genes Dev. 5:485-490: Hannon (2002) Nature 41: 244-251). In thetransfection of mammalian cells with long dsRNA, the activation ofprotein kinase R and RnaseL brings about unspecific effects, such as,for example, an interferon response (Stark et al. (1998) Annu. Rev.Biochem. 67: 227-264; He and Katze (2002) Viral Immunol. 15: 95-119).These unspecific effects are avoided when shorter, for example 21- to23-mer, so-called siRNA (small interfering RNA), is used, becauseunspecific effects are not triggered by siRNA that is shorter than 30 bp(Elbashir et al. (2001) Nature 411: 494-498).

The nucleic acid of the inventive polymeric carrier cargo complex maythus be a double-stranded RNA (dsRNA) having a length of from 17 to 29,preferably from 19 to 25, and preferably being at least 90%, morepreferably 95% and especially 100% (of the nucleotides of a dsRNA)complementary to a section of the nucleic acid sequence of a(therapeutically relevant) protein or antigen described (as activeingredient) hereinbefore, either a coding or a non-coding section,preferably a coding section. 90% complementary means that with a lengthof a dsRNA described herein of, for example, 20 nucleotides, thiscontains not more than 2 nucleotides without correspondingcomplementarity with the corresponding section of the mRNA. The sequenceof the double-stranded RNA used according to the invention as thenucleic acid of the inventive polymeric carrier cargo complex is,however, preferably wholly complementary in its general structure with asection of the nucleic acid of a therapeutically relevant protein orantigen described hereinbefore. In this context the nucleic acid of theinventive polymeric carrier cargo complex formed by the nucleic acidcargo and a polymeric carrier molecule according to generic formula (I)may be a dsRNA having the general structure 5′-(N₁₇₋₂₉)-3′, preferablyhaving the general structure 5′-(N₁₉₋₂₅)-3′, more preferably having thegeneral structure 5′-(N₁₉₋₂₄)-3′, or yet more preferably having thegeneral structure 5′-(N₂₁₋₂₃)-3′, wherein for each general structureeach N is a (preferably different) nucleotide of a section of the mRNAof a therapeutically relevant protein or antigen described hereinbefore,preferably being selected from a continuous number of 17 to 29nucleotides of the mRNA of a therapeutically relevant protein or antigenand being present in the general structure 5′-(N₁₋₂₉)-3′ in theirnatural order. In principle, all the sections having a length of from 17to 29, preferably from 19 to 25, base pairs that occur in the codingregion of the mRNA can serve as target sequence for a dsRNA herein.Equally, dsRNAs used as nucleic acid of the inventive polymeric carriercargo complex can also be directed against nucleotide sequences of a(therapeutically relevant) protein or antigen described (as activeingredient) hereinbefore that do not lie in the coding region, inparticular in the 5′ non-coding region of the mRNA, for example,therefore, against non-coding regions of the mRNA having a regulatoryfunction. The target sequence of the dsRNA used as nucleic acid of theinventive polymeric carrier cargo complex can therefore lie in thetranslated and untranslated region of the mRNA and/or in the region ofthe control elements of a protein or antigen described hereinbefore. Thetarget sequence of a dsRNA used as nucleic acid of the inventivepolymeric carrier cargo complex can also lie in the overlapping regionof untranslated and translated sequence; in particular, the targetsequence can comprise at least one nucleotide upstream of the starttriplet of the coding region of the mRNA.

Immunostimulatory Nucleic Acids: a) Immunostimulatory CpG Nucleic Acids:

According to another alternative, the nucleic acid of the inventivepolymeric carrier cargo complex formed by the nucleic acid cargo and apolymeric carrier molecule according to generic formula (I) (oraccording to any of its subformulas herein) may be in the form of a a(n)(immunostimulatory) CpG nucleic acid, in particular CpG-RNA or CpG-DNA,which preferably induces an innate immune response. A CpG-RNA or CpG-DNAused according to the invention can be a single-stranded CpG-DNA (ssCpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA(ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleicacid used according to the invention is preferably in the form ofCpG-RNA, more preferably in the form of single-stranded CpG-RNA (ssCpG-RNA). Also preferably, such CpG nucleic acids have a length asdescribed above. Preferably the CpG motifs are unmethylated.

b) Immunostimulatory RNA (isRNA):

Likewise, according to a further alternative, the nucleic acid of theinventive polymeric carrier cargo complex formed by the nucleic acidcargo and a polymeric carrier molecule according to generic formula (I)(or according to any of its subformulas herein) may be in the form of aof an immunostimulatory RNA (isRNA), which preferably elicits an innateimmune response. Such an immunostimulatory RNA may be any(double-stranded or single-stranded) RNA, e.g. a coding RNA, as definedherein. Preferably, the immunostimulatory RNA may be a single-stranded,a double-stranded or a partially double-stranded RNA, more preferably asingle-stranded RNA, and/or a circular or linear RNA, more preferably alinear RNA. More preferably, the immunostimulatory RNA may be a (linear)single-stranded RNA. Even more preferably, the immunostimulatory RNA maybe a (long) (linear) single-stranded) non-coding RNA. In this context itis particular preferred that the isRNA carries a triphosphate at its5′-end which is the case for in vitro transcribed RNA. Animmunostimulatory RNA may also occur as a short RNA oligonucleotide asdefined herein. An immunostimulatory RNA as used herein may furthermorebe selected from any class of RNA molecules, found in nature or beingprepared synthetically, and which can induce an innate immune responseand may support an adaptive immune response induced by an antigen. Inthis context, an immune response may occur in various ways. Asubstantial factor for a suitable (adaptive) immune response is thestimulation of different T-cell sub-populations. T-lymphocytes aretypically divided into two sub-populations, the T-helper 1 (Th1) cellsand the T-helper 2 (Th2) cells, with which the immune system is capableof destroying intracellular (Th1)) and extracellular (Th2) pathogens(e.g. antigens). The two Th cell populations differ in the pattern ofthe effector proteins (cytokines) produced by them. Thus, Th1 cellsassist the cellular immune response by activation of macrophages andcytotoxic T-cells. Th2 cells, on the other hand, promote the humoralimmune response by stimulation of B-cells for conversion into plasmacells and by formation of antibodies (e.g. against antigens). TheTh1/Th2 ratio is therefore of great importance in the induction andmaintenance of an adaptive immune response. In connection with thepresent invention, the Th1/Th2 ratio of the (adaptive) immune responseis preferably shifted in the direction towards the cellular response(Th1 response) and a cellular immune response is thereby induced.According to one example, the innate immune system which may support anadaptive immune response, may be activated by ligands of Toll-likereceptors (TLRs). TLRs are a family of highly conserved patternrecognition receptor (PRR) polypeptides that recognizepathogen-associated molecular patterns (PAMPs) and play a critical rolein innate immunity in mammals. Currently at least thirteen familymembers, designated TLR1 TLR13 (Toll-like receptors: TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13), havebeen identified. Furthermore, a number of specific TLR ligands have beenidentified. It was e.g. found that unmethylated bacterial DNA andsynthetic analogs thereof (CpG DNA) are ligands for TLR9 (Hemmi H et al.(2000) Nature 408:740-5; Bauer S et al. (2001) Proc NatlAcadSci USA 98,9237-42). Furthermore, it has been reported that ligands for certainTLRs include certain nucleic acid molecules and that certain types ofRNA are immunostimulatory in a sequence-independent orsequence-dependent manner, wherein these various immunostimulatory RNAsmay e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors suchas RIG-I, MDA-5, etc. E.g. Lipford et al. determined certainG,U-containing oligoribonucleotides as immunostimulatory by acting viaTLR7 and TLR8 (see WO 03/086280). The immunostimulatory G,U-containingoligoribonucleotides described by Lipford et al. were believed to bederivable from RNA sources including ribosomal RNA, transfer RNA,messenger RNA, and viral RNA.

The immunostimulatory RNA (isRNA) used as the nucleic acid molecule ofthe inventive polymeric carrier cargo complex formed by the nucleic acidcargo and a polymeric carrier molecule according to generic formula (I)(or according to any of its subformulas herein) may thus comprise anyRNA sequence known to be immunostimulatory, including, without beinglimited thereto, RNA sequences representing and/or encoding ligands ofTLRs, preferably selected from human family members TLR1 TLR10 or murinefamily members TLR1 TLR13, more preferably selected from (human) familymembers TLR1 TLR10, even more preferably from TLR7 and TLR8, ligands forintracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g.Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases:two parallel ways to trigger antiviral responses. Mol. Cell 22,561-569), or any other immunostimulatory RNA sequence. Furthermore,(classes of) immunostimulatory RNA molecules, used as the nucleic acidmolecule of the inventive polymeric carrier cargo complex may includeany other RNA capable of eliciting an immune response. Without beinglimited thereto, such an immunostimulatory RNA may include ribosomal RNA(rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA).Such an immunostimulatory RNA may comprise a length of 1000 to 5000, of500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to100, of 5 to 50 or of 5 to 30 nucleotides.

According to a particularly preferred aspect of this embodiment of thepresent invention, such immunostimulatory nucleic acid sequencesparticularly isRNA consist of or comprise a nucleic acid of formula(III) or (IV):

G_(l)XmGn  (formula (III))

wherein:

-   G is guanosine, uracil or an analogue of guanosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the above-mentioned nucleotides;-   I is an integer from 1 to 40,    -   wherein    -   when l=1 G is guanosine or an analogue thereof,    -   when l>1 at least 50% of the nucleotides are guanosine or an        analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3 X is uracil or an analogue thereof,    -   when m>3 at least 3 successive uracils or analogues of uracil        occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1 G is guanosine or an analogue thereof,    -   when n>1 at least 50% of the nucleotides are guanosine or an        analogue thereof.

C_(l)XmCn  (formula (IV))

wherein:

-   C is cytosine, uracil or an analogue of cytosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the above-mentioned nucleotides;-   l is an integer from 1 to 40,    -   wherein    -   when l=1 C is cytosine or an analogue thereof,    -   when l>1 at least 50% of the nucleotides are cytosine or an        analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3 X is uracil or an analogue thereof,    -   when m>3 at least 3 successive uracils or analogues of uracil        occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1 C is cytosine or an analogue thereof,    -   when n>1 at least 50% of the nucleotides are cytosine or an        analogue thereof.

The nucleic acids of formula (III) or (IV), which may be used as thenucleic acid cargo of the inventive polymeric carrier cargo complex maybe relatively short nucleic acid molecules with a typical length ofapproximately from 5 to 100 (but may also be longer than 100 nucleotidesfor specific embodiments, e.g. up to 200 nucleotides), from 5 to 90 orfrom 5 to 80 nucleotides, preferably a length of approximately from 5 to70, more preferably a length of approximately from 8 to 60 and, morepreferably a length of approximately from 15 to 60 nucleotides, morepreferably from 20 to 60, most preferably from 30 to 60 nucleotides. Ifthe nucleic acid of the inventive nucleic acid cargo complex has amaximum length of e.g. 100 nucleotides, m will typically be <=98. Thenumber of nucleotides G in the nucleic acid of formula (III) isdetermined by l or n. l and n, independently of one another, are each aninteger from 1 to 40, wherein when l or n=1 G is guanosine or ananalogue thereof, and when l or n>1 at least 50% of the nucleotides areguanosine or an analogue thereof. For example, without implying anylimitation, when l or n=4 G_(l) or G_(n) can be, for example, a GUGU,GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG or GGGG, etc.; when l orn=5 G_(l) or G_(n) can be, for example, a GGGUU, GGUGU, GUGGU, UGGGU,UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG, GGUGG, GUGGG, UGGGG, or GGGGG,etc.; etc. A nucleotide adjacent to X_(m) in the nucleic acid of formula(III) according to the invention is preferably not a uracil. Similarly,the number of nucleotides C in the nucleic acid of formula (IV)according to the invention is determined by l or n. l and n,independently of one another, are each an integer from 1 to 40, whereinwhen l or n=1 C is cytosine or an analogue thereof, and when l or n>1 atleast 50% of the nucleotides are cytosine or an analogue thereof. Forexample, without implying any limitation, when l or n=4, C_(l) or C_(n)can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC, CUCC,UCCC or CCCC, etc.; when l or n=5 C_(l) or C_(n) can be, for example, aCCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC, CUCUC, CCCCU, CCCUC,CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. A nucleotide adjacent to X_(m)in the nucleic acid of formula (IV) according to the invention ispreferably not a uracil. Preferably, for formula (III), when l or n>1,at least 60%, 70%, 80%, 90% or even 100% of the nucleotides areguanosine or an analogue thereof, as defined above. The remainingnucleotides to 100% (when guanosine constitutes less than 100% of thenucleotides) in the flanking sequences G_(l) and/or G_(n) are uracil oran analogue thereof, as defined hereinbefore. Also preferably, l and n,independently of one another, are each an integer from 2 to 30, morepreferably an integer from 2 to 20 and yet more preferably an integerfrom 2 to 15.

The lower limit of l or n can be varied if necessary and is at least 1,preferably at least 2, more preferably at least 3, 4, 5, 6, 7, 8, 9 or10. This definition applies correspondingly to formula (IV).

According to a further particularly preferred aspect of this embodiment,such immunostimulatory nucleic acid sequences particularly isRNA consistof or comprise a nucleic acid of formula (V) or (VI):

(N_(u)G_(l)X_(m)G_(n)N_(v))_(a),  (formula (V))

wherein:

-   G is guanosine (guanine), uridine (uracil) or an analogue of    guanosine (guanine) or uridine (uracil), preferably guanosine    (guanine) or an analogue thereof;-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine), or an analogue of these    nucleotides (nucleosides), preferably uridine (uracil) or an    analogue thereof;-   N is a nucleic acid sequence having a length of about 4 to 50,    preferably of about 4 to 40, more preferably of about 4 to 30 or 4    to 20 nucleic acids, each N independently being selected from    guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of these    nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein when l=1, G is guanosine (guanine) or an analogue        thereof, when l>1, at least 50% of these nucleotides        (nucleosides) are guanosine (guanine) or an analogue    -   thereof;-   m is an integer and is at least 3;    -   wherein when m=3, X is uridine (uracil) or an analogue thereof,        and        -   when m>3, at least 3 successive uridines (uracils) or            analogues of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein when n=1, G is guanosine (guanine) or an analogue        thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are guanosine (guanine) or an analogue        -   thereof;-   u,v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v≧1, or        -   when v=0, u≧1;            wherein the nucleic acid molecule of formula (V) has a            length of at least 50 nucleotides, preferably of at least            100 nucleotides, more preferably of at least 150            nucleotides, even more preferably of at least 200            nucleotides and most preferably of at least 250 nucleotides.

(N_(u)C_(l)X_(m)C_(n)N_(v))_(a)  (formula (VI))

wherein:

-   C is cytidine (cytosine), uridine (uracil) or an analogue of    cytidine (cytosine) or uridine (uracil), preferably cytidine    (cytosine) or an analogue thereof;-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of the    above-mentioned nucleotides (nucleosides), preferably uridine    (uracil) or an analogue thereof;-   N is each a nucleic acid sequence having independent from each other    a length of about 4 to 50, preferably of about 4 to 40, more    preferably of about 4 to 30 or 4 to 20 nucleic acids, each N    independently being selected from guanosine (guanine), uridine    (uracil), adenosine (adenine), thymidine (thymine), cytidine    (cytosine) or an analogue of these nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein when l=1, C is cytidine (cytosine) or an analogue        thereof,        -   when l>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue        -   thereof;-   m is an integer and is at least 3;    -   wherein when m=3, X is uridine (uracil) or an analogue thereof,        -   when m>3, at least 3 successive uridines (uracils) or            analogues of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein when n=1, C is cytidine (cytosine) or an analogue        thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue        -   thereof.-   u, v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v≧1, or        -   when v=0, u≧1;            wherein the nucleic acid molecule of formula (VI) according            to the invention has a length of at least 50 nucleotides,            preferably of at least 100 nucleotides, more preferably of            at least 150 nucleotides, even more preferably of at least            200 nucleotides and most preferably of at least 250            nucleotides.

For formula (VI), any of the definitions given above for elements N(i.e. N_(u) and N_(v)) and X (X_(m)), particularly the core structure asdefined above, as well as for integers a, l, m, n, u and v, similarlyapply to elements of formula (V) correspondingly, wherein in formula(VI) the core structure is defined by C_(l)X_(m)C_(n). The definition ofbordering elements N_(u) and N_(v) is identical to the definitions givenabove for N_(u) and N_(v).

According to a very particularly preferred aspect of this embodiment,the inventive nucleic acid molecule according to formula (V) may beselected from e.g. any of the following sequences:

(SEQ ID NO: 359)UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACG(SEQ ID NO: 360) UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUCCAAAAAAAAAAAAAAACCCACGCAAGGA UCUUCAUGUGC(SEQ ID NO: 361) GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACU CUAUUAGAUC(SEQ ID NO: 362) GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAG (SEQ ID NO: 363)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAGAGCUACGCAGGUUCGCAAUAAAAGCGUUGAUUAGUGUGCAUAGAACAGACCUCUUAUUCGGUGAAACGCCAGAAUGCUAAAUUCCAAUAACUCUUCCCAAAACGCGUACGGCCGAAGACGCGCGCUUAUCUUGUGUACGUUCUCGCACAUGGAAGAAUCAGCGGGCAUGGUGGUAGGGCAAUAGGGGAGCUGGGUAGCAGCGAAAAAGGGCCCCUGCGCACGUAGCUUCGCUGUUCGUCUGAAACAACCCGGCAUCCGUUGUAGCGAUCCCGUUAUCAGUGUUAUUCUUGUGCGCACUAAGAUUCAUGGUGUAGUCGACAAUAACAGCGUCUUGGCAGAUUCUGGUCACGUGCCCUAUGCCCGGGCUUGUGCCUCUCAGGUGCACAGCGAUACUUAAAGCCUUCAAGGUACUCGACGUGGGUACCGAUUCGUGACACUUCCUAAGAUUAUUCCACUGUGUUAGCCCCGCACCGCCGACCUAAACUGGUCCAAUGUAUACGCAUUCGCUGAGCGGAUCGAUAAUAAAAGCUUGAAUU (SEQ ID NO: 364)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGA UGCUGGCCCAGAUC(R 722 SEQ ID NO: 365)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAG GUCUGCUCUA(SEQ ID NO: 366) GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAGAACGAACUGACCUGACGCCUGAACUUAUGAGCGUGCGUAUUUUUUUUUUUUUUUUUUUUUUUCCUCCCAACAAAUGUCGAUCAAUAGCUGGGCUGUUGGAGACGCGUCAGCAAAUGCCGUGGCUCCAUAGGACGUGUAGACUUCUAUUUUUUUUUUUUUUUUUUUUUCCCGGGACCACAAAUAAUAUUCUUGCUUGGUUGGGCGCAAGGGCCCCGUAUCAGGUCAUAAACGGGUACAUGUUGCACAGGCUCCUUUUUUUUUUUUUUUUUUUUUUUCGCUGAGUUAUUCCGGUCUCAAAAGACGGCAGACGUCAGUCGACAACACGGUCUAAAGCAGUGCUACAAUCUGCCGUGUUCGUGUUUUUUUUUUUUUUUUUUUUGUGAACCUACACGGCGUGCACUGUAGUUCGCAAUUCAUAGGGUACCGGCUCAGAGUUAUGCCUUGGUUGAAAACUGCCCAGCAUACUUUUUUUUUUUUUUUUUUUUCAUAUUCCCAUGCUAAGCAAGGGAUGCCGCGAGUCAUGU UAAGCUUGAAUU

According to another very particularly preferred embodiment, the nucleicacid molecule according to formula (VI) may be selected from e.g. any ofthe following sequences:

(SEQ ID NO: 367) UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACG or (SEQ ID NO: 368)UAGCGAAGCUCUUGGACCUACCUUUUUUUU UUUUUUUCCCUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUGGAAAAAAAA AAAAAAAGGGACGCAAGGAUCUUCAUGUGC

In a further preferred embodiment the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may also occurin the form of a modified nucleic acid.

According to a further aspect, the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex may be provided as a“stabilized nucleic acid”, preferably as a stabilized RNA or DNA, morepreferably as a RNA that is essentially resistant to in vivo degradation(e.g. by an exo- or endo-nuclease).

In this context, the nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex may contain backbonemodifications, sugar modifications or base modifications. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained in thenucleic acid molecule of the inventive polymeric carrier cargo complexare chemically modified. A sugar modification in connection with thepresent invention is a chemical modification of the sugar of thenucleotides of the nucleic acid molecule of the inventive polymericcarrier cargo complex. Furthermore, a base modification in connectionwith the present invention is a chemical modification of the base moietyof the nucleotides of the nucleic acid molecule of the inventivepolymeric carrier cargo complex.

According to a further aspect, the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex can contain a lipidmodification.

The nucleic acid of the inventive polymeric carrier cargo complex asdefined herein may also be in the form of a modified nucleic acid,wherein any modification, as defined herein, may be introduced into thenucleic acid. Modifications as defined herein preferably lead to afurther stabilized nucleic acid.

According to one aspect, the nucleic acid of the inventive polymericcarrier cargo complex as defined herein may thus be provided as a“stabilized nucleic acid”, preferably as a stabilized mRNA, morepreferably as an mRNA that is essentially resistant to in vivodegradation (e.g. by an exo- or endo-nuclease). Such stabilization canbe effected, for example, by a modified phosphate in which phosphates ofthe backbone of the nucleotides contained in the nucleic acid arechemically modified. The nucleic acid of the inventive polymeric carriercargo complex may additionally or alternatively also contain sugar orbase modifications. The nucleic acid of the inventive polymeric carriercargo complex, particularly if provided as an mRNA, can also bestabilized against degradation by RNases by the addition of a so-called“5′ cap” structure. Particular preference is given in this connection toan m7G(5′)ppp (5′(A,G(5′)ppp(5′)A or G(5′)ppp(5′)G as the 5′ cap″structure. According to a further aspect, the nucleic acid of theinventive polymeric carrier cargo complex may contain, especially if thenucleic acid is in the form of an mRNA, a poly-A tail on the 3′ terminusof typically about 10 to 200 adenosine nucleotides, preferably about 10to 100 adenosine nucleotides, more preferably about 20 to 100 adenosinenucleotides or even more preferably about 40 to 80 adenosinenucleotides. According to a further aspect, the nucleic acid of theinventive polymeric carrier cargo complex may contain, especially if thenucleic acid is in the form of an mRNA, a poly-C tail on the 3′ terminusof typically about 10 to 200 cytosine nucleotides, preferably about 10to 100 cytosine nucleotides, more preferably about 20 to 70 cytosinenucleotides or even more preferably about 20 to 60 or even 10 to 40cytosine nucleotides. According to another aspect, the nucleic acid ofthe inventive polymeric carrier cargo complex may be modified, and thusstabilized, especially if the nucleic acid is in the form of an mRNA, bymodifying the G/C content of the nucleic acid, particularly an mRNA,preferably of the coding region thereof.

In a particularly preferred aspect of the present invention, the G/Ccontent of the coding region of the nucleic acid of the inventivepolymeric carrier cargo complex, especially if the nucleic acid is inthe form of an mRNA, is modified, particularly increased, compared tothe G/C content of the coding region of its particular wild-type mRNA,i.e. the unmodified mRNA. The encoded amino acid sequence of the atleast one mRNA is preferably not modified compared to the coded aminoacid sequence of the particular wild-type mRNA. Preferably, the G/Ccontent of the coding region of nucleic acid of the inventive polymericcarrier cargo complex, especially if the nucleic acid is in the form ofan mRNA, is increased by at least 7%, more preferably by at least 15%,particularly preferably by at least 20%, compared to the G/C content ofthe coded region of the wild-type mRNA which codes for an antigen,antigenic protein or antigenic peptide as determined herein or itsfragment or variant thereof. According to a specific embodiment at least5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, evenmore preferably at least 80% and most preferably at least 90%, 95% oreven 100% of the substitutable codons in the region coding for a proteinor peptide as defined herein or its fragment or variant thereof or thewhole sequence of the wild type mRNA sequence are substituted, therebyincreasing the GC/content of said sequence. In this context, it isparticularly preferable to increase the G/C content of the nucleic acidof the inventive polymeric carrier cargo complex, especially if thenucleic acid is in the form of an mRNA, to the maximum (i.e. 100% of thesubstitutable codons), in particular in the region coding for a protein,compared to the wild-type sequence. According to the invention, afurther preferred modification of the nucleic acid of the inventivepolymeric carrier cargo complex, especially if the nucleic acid is inthe form of an mRNA, the region which codes for the adjuvant protein ismodified compared to the corresponding region of the wild-type mRNA suchthat at least one codon of the wild-type sequence which codes for a tRNAwhich is relatively rare in the cell is exchanged for a codon whichcodes for a tRNA which is relatively frequent in the cell and carriesthe same amino acid as the relatively rare tRNA. By this modification,the sequences of the nucleic acid, especially if the nucleic acid is inthe form of an mRNA, is modified such that codons for which frequentlyoccurring tRNAs are available are inserted. In other words, according tothe invention, by this modification all codons of the wild-type sequencewhich code for a tRNA which is relatively rare in the cell can in eachcase be exchanged for a codon which codes for a tRNA which is relativelyfrequent in the cell and which, in each case, carries the same aminoacid as the relatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

Nucleic acid molecules used according to the present invention asdefined herein may be prepared using any method known in the art,including synthetic methods such as e.g. solid phase synthesis, as wellas in vitro methods, such as in vitro transcription reactions or in vivoreactions, such as in vivo propagation of DNA plasmids in bacteria.

According to another particularly preferred embodiment, the nucleic acidof the inventive polymeric carrier cargo complex, especially if thenucleic acid is in the form of a coding nucleic acid, preferably anmRNA, may additionally or alternatively encode a secretory signalpeptide. Such signal peptides are sequences, which typically exhibit alength of about 15 to 30 amino acids and are preferably located at theN-terminus of the encoded peptide, without being limited thereto. Signalpeptides as defined herein preferably allow the transport of the proteinor peptide as encoded by the nucleic acid of the present invention,especially if the nucleic acid is in the form of an mRNA, into a definedcellular compartiment, preferably the cell surface, the endoplasmicreticulum (ER) or the endosomal-lysosomal compartiment.

Any of the above modifications may be applied to the nucleic acid of theinventive polymeric carrier cargo complex, especially if the nucleicacid is in the form of an mRNA, and further to any nucleic acid as usedin the context of the present invention and may be, if suitable ornecessary, be combined with each other in any combination, provided,these combinations of modifications do not interfere with each other inthe respective nucleic acid. A person skilled in the art will be able totake his choice accordingly.

Proteins or peptides as encoded by the nucleic acid of the inventivepolymeric carrier cargo complex as defined herein, may comprisefragments or variants of those sequences. Additionally, the nucleic acidof the inventive polymeric carrier cargo complex may comprise fragmentsor variants of those coding sequences. Such fragments or variants maytypically comprise a sequence having a sequence identity with one of theabove mentioned proteins or peptides or sequences of their encodingnucleic acid sequences of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,preferably at least 70%, more preferably at least 80%, equally morepreferably at least 85%, even more preferably at least 90% and mostpreferably at least 95% or even 97%, to the entire wild-type sequence,either on nucleic acid level or on amino acid level.

“Fragments” of proteins or peptides in the context of the presentinvention may comprise a sequence of an protein or peptide as definedherein, which is, with regard to its amino acid sequence (or its encodednucleic acid sequence), N-terminally, C-terminally and/orintrasequentially truncated compared to the amino acid sequence of theoriginal (native) protein (or its encoded nucleic acid sequence). Suchtruncation may thus occur either on the amino acid level orcorrespondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid sequence of such a protein or peptide. Thesame applies accordingly to nucleic acids.

Such fragments of proteins or peptides in the context of the presentinvention may furthermore comprise a sequence of a protein or peptide asdefined herein, which has a length of about 6 to about 20 or even moreamino acids, e.g. fragments as processed and presented by MHC class Imolecules, preferably having a length of about 8 to about 10 aminoacids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino acids), orfragments as processed and presented by MHC class II molecules,preferably having a length of about 13 or more amino acids, e.g. 13, 14,15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragmentsmay be selected from any part of the amino acid sequence. Thesefragments are typically recognized by T-cells in form of a complexconsisting of the peptide fragment and an MHC molecule, i.e. thefragments are typically not recognized in their native form.

The fragments of proteins or peptides as defined herein may alsocomprise epitopes of those proteins or peptides. Epitopes (also called“antigen determinants”) in the context of the present invention aretypically fragments located on the outer surface of (native) proteins orpeptides as defined herein, preferably having 5 to 15 amino acids, morepreferably having 5 to 12 amino acids, even more preferably having 6 to9 amino acids, which may be recognized by antibodies or B-cellreceptors, i.e. in their native form. Such epitopes of proteins orpeptides may furthermore be selected from any of the herein mentionedvariants of such proteins or peptides. In this context antigenicdeterminants can be conformational or discontinous epitopes which arecomposed of segments of the proteins or peptides as defined herein thatare discontinuous in the amino acid sequence of the proteins or peptidesas defined herein but are brought together in the three-dimensionalstructure or continuous or linear epitopes which are composed of asingle polypeptide chain.

“Variants” of proteins or peptides as defined herein may be encoded bythe nucleic acid of the inventive polymeric carrier cargo complex,wherein nucleotides of the nucleic acid, encoding the protein or peptideas defined herein, are exchanged. Thereby, a protein or peptide may begenerated, having an amino acid sequence which differs from the originalsequence in one or more mutation(s), such as one or more substituted,inserted and/or deleted amino acid(s). Preferably, these fragmentsand/or variants have the same biological function or specific activitycompared to the full-length native potein, e.g. its specific antigenicproperty.

The present invention also provides a method of preparing the inventivepolymeric carrier molecule according to formula (I)L-P¹—S—[S—P²—S]_(n)—S—P³-L as defined herein or according to anysubformula thereof as defined herein (e.g. (Ia), etc.). It also providesthe product obtained or obtainable by such an inventive method (productby process). The method preferably comprises following steps:

-   -   a) providing at least one cationic or polycationic protein or        peptide as component P² as defined herein and/or at least one        cationic or polycationic polymer as component P² as defined        herein, and optionally at least one further component (e.g.        (AA)_(x), [(AA_(x))]_(z), etc.)., preferably in the ratios        indicated above by formula (I), mixing these components,        preferably in a basic milieu as defined herein, preferably in        the presence of oxygen or a further starter as defined herein        which leads to mild oxidation conditions, preferably at a pH, at        a temperature and at time as defined herein, and thereby        condensing and thus polymerizing these components with each        other via disulfide bonds (in a polymerization condensation or        polycondensation) to obtain a repetitive component        H—[S—P²—S]_(n)—H or H{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}H, etc.;

-   b) providing a hydrophilic polymer P¹ and/or P³ as defined herein,    optionally modified with a ligand L and/or an amino acid component    (AA)_(x) as defined herein;

-   c) mixing the hydrophilic polymer P¹ and/or P³ provided according to    step b) with the repetitive component H—[S—P²—S]_(n)—H or    H{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}H, etc. obtained according to step    a), typically in a ratio of about 2:1, (and thereby typically    terminating the polymerization condensation or polycondensation    reaction) and obtaining the inventive polymeric carrier, preferably    according to formula (I) as defined herein or according to any    subformula thereof as defined herein;

-   d) optionally purifying the inventive polymeric carrier obtained    according to step c), preferably using a method as defined herein;

-   e) optionally adding a nucleic acid as defined herein to the    inventive polymeric carrier obtained according to step c) or d),    preferably in the above mentioned ratios, and complexing the nucleic    acid with the polymeric carrier obtained according to step c) or d)    to obtain an inventive polymeric carrier cargo complex as defined    herein.

The inventive method of preparing the inventive polymeric carrieraccording to formula (I) as defined herein represents a multi-stepcondensation polymerization or polycondensation reaction via —SHmoieties of the educts, e.g. component(s) P² as defined herein, furthercomponents P¹ and/or P³ and optionally further components (AA)_(x). Thecondensation polymerization or polycondensation reaction preferablyleads to the inventive polymeric carrier as a condensation polymer,wherein the single components are linked by disulfide bonds. Thiscondensation polymerization leads to the inventive polymeric carrieraccording to formula (I) preparing in a first step a) of thecondensation reaction the inventive repetitive componentH—[S—P²—S]_(n)—H or a variant thereof as a sort of a “core” or “centralmotif” of the inventive polymeric carrier. In a second step b)components P¹ and/or P³ are provided, which allow to terminate or tosomehow “coat” the inventive repetitive component H—[S—P²—S]_(n)—H or avariant thereof in a third step c) by adding components P¹ and/or P³ asdefined herein (optionally modified with a ligand L and/or an amino acidcomponent (AA)_(x) as defined herein) to the condensation productobtained according to step a). In subsequent step d), this product maybe purified and further used to complex a nucleic acid cargo as definedherein to obtain an inventive complex.

It is important to understand that the inventive method is based on anequibrility reaction under mild oxidation conditions in steps a), (b))and c), which, upon balancing the equilibirity state, allows to obtainthe inventive polymeric carrier according to formula (I) above oraccording to any of its subformulas comprising the selected componentsin the desired molar ratios. For this purpose, long reaction times areenvisaged to achieve an equibrility state in steps a), (b)) and c). Iffor example a condensation polymerization is to be carried out using amolar ratio of 5 components P² in step a), the equilibrium issurprisingly settled at a polymer length of about 5 after sufficienttime, preferably e.g. >12 hours. However, due to the equilibrium thepolymer length (as defined by n) is not fixed at a specific value, e.g.5, but may vary accordingly within the equibrility reaction.Accordingly, about 5 may mean about 4 to 6, or even about 3 to 7.Preferably, the polymer length and thus the integer n (and thus a, b anda+b) varies within a limit of about ±1, or ±2.

As defined herein in a step a) of the inventive method of preparing theinventive polymeric carrier according to formula (I) at least onecationic or polycationic protein or peptide as component P² as definedherein and/or at least one cationic or polycationic polymer as componentP² as defined herein are provided, preferably in the ratios indicatedabove by formula (I). These components are mixed, preferably in a basicmilieu as defined herein, preferably in the presence of oxygen or afurther starter as defined herein which leads to mild oxidationconditions, preferably at a pH, and at a temperature and at a time asdefined herein, and thereby condensing and thus polymerizing thesecomponents with each other via disulfide bonds (in a polymerizationcondensation or polycondensation) to obtain a repetitive componentH—[S—P²—S]_(n)—H.

According to an alternative, in step a) of the inventive method ofpreparing the inventive polymeric carrier at least one cationic orpolycationic protein or peptide and/or at least one cationic orpolycationic polymer are provided and used as component(s) P² as definedherein, and additionally at least one amino acid component (AA)_(x) isprovided as defined herein, and components P² and (AA)_(x) are used fora polymerization condensation or polycondensation according to step a).Preferably, the components are all provided in the ratios indicatedabove by formula (Ia), mixed, preferably in a basic milieu as definedherein, preferably in the presence of oxygen or a further starter asdefined herein which leads to mild oxidation conditions, preferably at apH, at a temperature and at time as defined herein. Upon mixing andstarting the reaction, the components are condensed and thus polymerizedwith each other via disulfide bonds (in a polymerization condensation orpolycondensation) to obtain a repetitive componentH—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—H.

In both of the above alternatives, different component(s) P²,particularly different peptides and/or different polymers as componentP², may be selected in the condensation polymerization as indicatedabove. In this context, the selection of different component(s) P² istypically dependent upon the desired properties of the final inventivepolymeric carrier and the desired cationic strength of the finalinventive polymeric carrier or its central core motif. Accordingly, therepetitive component [S—P²—S]_(n), may furthermore be “diluted” ormodified in the above alternative of step a) e.g. by introducing anamino acid component (AA)_(x) as defined herein, preferably in the abovedefined ratios. Thereby, a modified central core motif{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} may be obtained, wherein the cationiccharacter of (unmodified) repetitive component [S—P²—S]_(n) typicallyremains in the limitations as defined herein. The properties of thefinal inventive polymeric carrier may thus be adjusted as desired withproperties of components (AA)_(x) by inserting amino acid component(AA)_(x) as defined herein in steps a), b) and/or c).

In all cases, step a) is based on an equibrility reaction under mildoxidation conditions which, upon balancing the equilibirity state,allows to obtain either inventive repetitive component H—[S—P²—S]_(n)—Hor inventive repetitive component H—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—Hin the desired molar ratios. In the equilibrity state, n is preferably1, 2, 3, 4, or 5 to 10, more preferably 4 to 9, and a+b=n is as definedabove, preferably a+b=1, 2, 3, 4, or 5 to 10, more preferably 4 to 9.For this purpose, long reaction times are envisaged to achieve anequibrility state in step a), most preferably e.g. >12 hours.Accordingly, step a) of the inventive method of preparing a polymericcarrier typically requires at least about 5 hours, even more preferablyat least about 7.5 hours or even 10 hours, most preferably at leastabout 12 hours, e.g. a reaction time of about 12 to 60 hours, a reactiontime of about 12 to 48 hours, a reaction time of about 12 to 36 hours,or a reaction time of about 12 to 24 hours, etc, wherein the lowerborder of 12 hours of thelatter ranges may also be adjusted to 10, 7.5,or even 5 hours. Advantageously, the equilibirity state can be balancedusing the inventive method.

In step a), the at least one cationic or polycationic protein or peptideas component P² as defined herein and/or at least one cationic orpolycationic polymer as component P² as defined herein, and optionallyat least one amino acid component (AA)_(x) as defined herein, arepreferably contained in a basic milieu in the step a) of the inventivemethod of preparing the inventive polymeric carrier according to formula(I) (or any of its subformulas, e.g. (Ia)). Such a basic milieutypically exhibits a pH range of about 6 to about 12, preferably a pHrange of about 7 to about 10, more preferably a pH range of about 8 toabout 10, e.g. about 8, 8.5, 9, 9.5, or 10 or any range selected fromany two of these or the aforementioned values.

Furthermore, the temperature of the solution in step a) is preferably ina range of about 5° C. to about 60° C., more preferably in a range ofabout 15° C. to about 40° C., even more preferably in a range of about20° C. to about 30° C., and most preferably in a range of about 20° C.to about 25° C., e.g. about 25° C.

In step a) of the inventive method of preparing the inventive polymericcarrier according to formula (I) (or any of its subformulas, e.g. (Ia))as defined herein buffers may be used as suitable. Preferred buffers maycomprise, but are not limited to carbonate buffers, borate buffers,Bicine buffer, CHES buffer, CAPS buffer, Ethanolamine containingbuffers, HEPES, MOPS buffer, Phosphate buffer, PIPES buffer, Trisbuffer, Tricine buffer, TAPS buffer, and/or TES buffer as bufferingagents. Particularly preferred is a carbonate buffer.

Upon mixing the components, preferably in the presence of oxygen,preferably in the presence of a basic mileu as defined herein, thecondensation polymerization or polycondensation reaction is started. Forthis purpose, the mixture in step a) is preferably exposed to oxygen ormay be started using a further starter, e.g. a catalytical amount of anoxidizing agent, e.g. DMSO, etc. To determine the desired polymer chainlength the condensation reaction has to be carried out under mildoxidation conditions, preferably in the presence of less than 30% DMSO,more preferably in the presence of less than 20% DMSO and mostpreferably in the presence of less than 10% DMSO. Upon start of thecondensation polymerization or polycondensation reaction the at leastone cationic or polycationic protein or peptide and/or at least onecationic or polycationic polymer as component P² and optionally at leastone amino acid component (AA)_(x) as defined herein, are condensed andthus polymerized with each other via disulfide bonds (polymerizationcondensation or polycondensation). In this reaction step a) preferablylinear polymers are created using monomers with at least two reactive—SH moieties, i.e. at least one cationic or polycationic protein orpeptide and/or at least one cationic or polycationic polymer ascomponent P² as defined herein, each component P² exhibiting at leasttwo free —SH-moieties as defined herein, e.g. at their terminal ends.However, components P² with more than two free —SH-moieties may be used,which may lead to branched polymers.

According to one other specific embodiment, the condensation productobtained according to step a) may be modified (e.g. in a step a1)) byadding an amino acid component (AA)_(x) or a mixed repetitive amino acidcomponent [(AA)_(x)]_(z) as defined herein e.g. to the terminal ends ofthe condensation product of step a). This may occur via anyfunctionality as defined herein, e.g. a —SH moiety or any furtherfunctionality described herein, preferably a —SH moiety. For thispurpose, amino acid component (AA)_(x) or a mixed repetitive amino acidcomponent [(AA)_(x)]_(z) may be provided with two (or even more)—SH-moieties, e.g. in a form represented by formulae “H(S-AA-S)_(x)H” or“H[S-(AA)_(x)-S]_(z)H”. Then, a polycondensation reaction may be carriedout with the products of step a), i.e. inventive repetitive componentH—[S—P²—S]_(n)—H or inventive repetitive componentH—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—H, leading to intermediate components

H(S-AA-S)_(x)—[S—P²—S]_(n)—(S-AA-S)_(x)H, or

H[S-(AA)_(x)-S]_(z)—[S—P²—S]_(n)—[S-(AA)_(x)-S]_(z)H, or

H(S-AA-S)_(x)—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—(S-AA-S)_(x)H, or

H[S-(AA)_(x)-S]_(z)—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—[S-(AA)_(x)-S]_(z)H.

Any single or all of these intermediate components or the inventiverepetitive component

H—[S—P²—S]_(n)—H

or the inventive repetitive component

H—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—H

obtained according to step a), may be used to be coupled to the polymersprovided in step b) of the inventive method.

According to a second step b) of the inventive method of preparing theinventive polymeric carrier according to formula (I) as defined herein(or according to any of its subformulas), a hydrophilic polymer P¹and/or P³ as defined herein is added to the condensation productobtained according to step a). In this context, the hydrophilic polymersP¹ and/or P³ as defined herein, preferably exhibit at least one—SH-moiety, more preferably only one —SH-moiety per hydrophilic polymersP¹ and/or P³ as defined herein, thereby terminally stopping thepolymerization condensation or polycondensation according to step a) instep c). Hydrophilic polymers P¹ and/or P³ as defined herein may be thesame or different, wherein these polymers may be selected according tothe desired properties. Typically, hydrophilic polymers P¹ and/or P³ asa whole may be added to the condensation product obtained according tostep a) in a ratio of about 2:1 hydrophilic polymer P¹ and/orP³:condensation product obtained according to step a).

According to one alternative, the hydrophilic polymer(s) P¹ and/or P³additionally may be modified with either a component L (ligand) asdefined herein or a component (AA)_(x) or [(AA)_(x)]_(z) as definedherein or both a component L (ligand) as defined herein and a component(AA)_(x) or [(AA)_(x)]_(z) as defined herein.

According to a first example, a ligand is attached to component(s) P¹and/or P³ as component L prior to providing component(s) P¹ and/or P³ instep b) via any functionality as defined herein, e.g. a —SH moiety. Thisligand is preferably attached to the hydrophilic polymer(s) P¹ and/or P³at one terminus of these polymers. If the attachment is carried out via—SH bonds, the hydrophilic polymer(s) P¹ and/or P³ are preferablyprovided with two (or even more) —SH-moieties., e.g. in a formrepresented by formulae HS—P¹—SH or HS—P³—SH. Ligand L preferablycarries only one —SH moiety. In this case, one —SH moiety of hydrophilicpolymer(s) P¹ and/or P³ is preferably protected in a first step using aprotecting group as known in the art. Then, the hydrophilic polymer(s)P¹ and/or P³ may be bound to a component L to form a first disulfidebond via the non-protected —SH moiety. The protected —SH-moiety ofhydrophilic polymer(s) P¹ and/or P³ is then typically deprotected forfurther reactions. This preferably leads to following intermediatecomponents

L-S—S—P¹—SH, or

HS—P³—S—S-L.

Alternatively, the above intermediate components may be providedsimilarly without the necessity of blocking the free —SH-moieties. Theseintermediate components may be used in step c) to be coupled with thecondensation products obtained according to step a) above, e.g. to forma second disulfide bond with inventive repetitive componentH—[S—P²—S]_(n)—H or inventive mixed repetitive componentH—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—H obtained according to step a) orany of its modifications, e.g. according to step a1). If the attachmentis carried out via other moieties, any of the reactions as definedherein may be used accordingly.

According to a further example, an amino acid component (AA)_(x) or amixed repetitive amino acid component [(AA)_(x)]_(z) as defined hereinmay be attached to component(s) P¹ and/or P³ prior to providingcomponent(s) P¹ and/or P³ via any functionality as defined herein, e.g.a —SH moiety. The amino acid component (AA)_(x) or a mixed repetitiveamino acid component [(AA)_(x)]_(z), may be attached to the hydrophilicpolymer(s) P¹ and/or P³ at any position within these polymers or at oneor both termini of these polymers. In one specific case, the amino acidcomponent (AA)_(x) or a mixed repetitive amino acid component[(AA)_(x)]_(z) may be provided as a linker between component(s) P¹and/or P³ and the condensation product obtained according to step a)above or as a linker between component(s) P¹ and/or P³ and a furthercomponent, e.g. a linker L, or according to another alternative, as aterminating component at one terminus of component(s) P¹ and/or P³. Inany of these cases, the attachment preferably may carried out via —SHbonds, wherein the hydrophilic polymer(s) P¹ and/or P³ are preferablyprovided with two (or even more) —SH-moieties., e.g. in a formrepresented by formula “HS—P¹—SH” or “HS—P³—SH”, wherein preferably oneof these to —SH moieties is protected, e.g. in a form represented byformula “HS—P¹—S-protecting group” or “protecting group-S—P³—SH”.Furthermore, amino acid component (AA)_(x) or a mixed repetitive aminoacid component [(AA)_(x)]_(z) are also preferably provided with two (oreven more) —SH-moieties, e.g. in a form represented by formulaeH(S-AA-S))_(x)—H″ or “H[S-(AA)_(x)—S]_(z)H”, wherein preferably one ofthese to —SH moieties is protected, e.g. in a form represented byformulae “protecting group-(S-AA-S)_(x)—SH” or“H[S-(AA)_(x)-S]_(z)-protecting group”. Then, a polycondensationreaction may be carried out with polymers “HS—P¹—S-protecting group” or“protecting group-S—P³—SH” leading to intermediate components

-   -   “protecting group-S—P¹—S—(S-AA-S)—S-protecting group”,    -   “protecting group-(S-AA-S)_(x)—S—S—P³—S-protecting group”,    -   “protecting group-S—P¹—S—[S-(AA)_(x)-S]_(z)-protecting group”,        or    -   “protecting group-[S-(AA)_(x)-S]_(z)—S—P³—S-protecting group”.

Any single or all of these intermediate components may then be used instep c) of the inventive method to be coupled to the condensationproduct according to step a).

For this purpose, at least one or both protecting groups (selected uponthe desired direction of the component in the final carrier) of eachintermediate compound may be deprotected prior to providing them in stepb), leading to following intermediate components

“HS—P¹—S—(S-AA-S)_(x)—SH”,

“H(S-AA-S))_(x)—S—S—P³—SH”,

“HS—P¹—S—[S-(AA)_(x)S]_(z)H”, or

“H[S-(AA)_(x)-S]_(z)—S—P³—SH”,

Alternatively, the above intermediate components may be providedsimilarly without the necessity of blocking the free —SH-moieties. Anysingle or all of these intermediate components may then be provided instep b) of the inventive method to be coupled to the condensationproduct according to step a).

If any of the afore mentioned intermediate components is provided instep b), this condensation reaction may be terminated in a step c) byadding a linker component as defined herein with one —SH-moiety (e.g.L-SH) or any further component with a single —SH moiety, e.g. as definedherein. In one further specific case, the amino acid component (AA)_(x)or a mixed repetitive amino acid component [(AA)_(x)]_(z) may be used asa terminal component at one terminus of component(s) P¹ and/or P³without adding a further component to the amino acid component (AA)_(x)or a mixed repetitive amino acid component [(AA)_(x)]_(z).

According to a further example, an amino acid component (AA)_(x) or amixed repetitive amino acid component [(AA)_(x)]_(z) as defined hereinmay be attached to component(s) P¹ and/or P³ prior to step c), whereincomponent(s) P¹ and/or P³ have been already modified with a linker. Forthis purpose, component(s) P¹ and/or P³ preferably carry (at least) two—SH moieties as defined herein, wherein a polycondensation is carriedout with a linker, carrying e.g. one —SH moiety. This reaction may becarried out by using protecting groups as defined herein, or,preferably, without protecting groups. Alternatively, any furtherfunctionality as defined herein except —SH moieties may be used forcoupling. Then, the second —SH moiety of component(s) P¹ and/or P³ maybe used to couple an amino acid component (AA)_(x) or a mixed repetitiveamino acid component MAW, as defined herein via —SH-moieties, e.g. in aform represented by formulae “H(S-AA-S)_(x)—H” or“H[S-(AA)_(x)-S]_(z)H”. The reaction preferably leads to followingintermediate compounds

“L-S—S—P¹—S—(S-AA-S)_(x)—SH”,

“L-S—S—P¹—S—[S-(AA)_(x)-S]_(z)H”, or

“HS—(S-AA-S)_(x)—S—S—P³—S—S-L”, or

“HS—[S-(AA)_(x)-S]_(z)—S—P³—S—S-L”;

or, if component L has been linked without a disulfide bond to followingintermediate products

“L-P¹—S—(S-AA-S)_(x)—SH”,

“L-P¹—S—[S-(AA)_(x)-S]_(z)H”, or

“HS—(S-AA-S)_(x)—S—S—P³³-L”, or

“HS—[S-(AA)_(x)-S]_(z)—S—P³-L”;

In step c) the hydrophilic polymers P¹ and/or P³ (or any of theintermediate components provided according to step b)) as definedherein, are provided and mixed with the repetitive componentH—[S—P²—S]_(n)—H, with the mixed repetitive componentH—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—H, or any of the intermediatecomponents obtained according to step a), typically in a ratio of about2:1. The reaction is typically started and carried out under conditionsalready described above for step a) (pH, temperature, reaction time,buffers, etc.). Step c) allows to terminate the polymerizationcondensation or polycondensation reaction and to obtain the inventivepolymeric carrier according to formula (I) or (Ia) or according to anyof subformulas thereof as defined herein, preferably the inventivepolymeric carrier according to formula (I)

L-P¹—S—[S—P²—S]_(n)—S—P³-L

or according to formula (Ia)

L-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-L.

According to a further step d) of the inventive method of preparing theinventive polymeric carrier according to formula (I) or (Ia) as definedherein, or according to any of subformulas thereof as defined herein,the inventive polymeric carrier obtained according to step c) isoptionally purified. Purification may occur by using chromatographicmethods, such as HPLC, FPLC, GPS, dialysis, etc.

According to a final step e) of the inventive method of preparing theinventive polymeric carrier according to formula (I) or (Ia) as definedherein, or according to any of subformulas thereof as defined herein, anucleic acid as defined herein is optionally added to the inventivepolymeric carrier obtained according to step c) or d), preferably in theabove mentioned ratios. Typically, in the inventive complex, thepolymeric carrier molecule according to generic formula (I) or (Ia) oraccording to any of subformulas thereof as defined herein, as definedherein and the nucleic acid are provided in a molar ratio of about 5 to10000, preferably in a molar ratio of about 5 to 5000, more preferablyin a molar ratio of about 5 to 2500, even more preferably in a molarratio of about 5 to 1000 polymeric carrier:nucleic acid, e.g. in a molarratio of about 10 to 10000, in a molar ratio of about 10 to 5000, in amolar ratio of about 25 to 2500, or in a molar ratio of about 50 to 1000polymeric carrier:nucleic acid. The N/P ratios are preferably asindicated above.

The inventive method of preparing the inventive polymeric carrieraccording to formula (I) or (Ia) or according to any of subformulasthereof as defined herein is particularly suitable to adapt the chemicalproperties of the desired inventive polymeric carrier due to specificselection of its components P², L, (AA)_(x), or [(AA)_(x)]_(z) therebyavoiding agglomeration and toxicity in vivo.

Furthermore, a skilled person would not have expected to obtain aninventive polymeric carrier using the above inventive method as theskilled person would always have expected that the polymer obtainedaccording to the inventive method due to general rules of equilibrityreactions leads to a monomeric content of component P², flanked bymonomeric components P¹ and/or P³, wherein the linkages are formed bydisulfide bonds. In contrast, the present inventors were surprisinglyable to show that when using a specific ratio of polymers and methodsteps as defined herein, particularly mild oxidation conditions duringthe polymerization reaction, the polymerization condensation can bedirected to specifically obtain a desired distribution of polymers and adesired average length and the desired inventive polymeric carrieraccording to generic formula (I) or (Ia) or according to any ofsubformulas thereof as defined herein without the necessity of blockingthe free —SH-moieties. This was not expected by a skilled person.

According to a further embodiment, the present invention also provides apharmaceutical composition, comprising the inventive polymeric carriercargo complex formed by a nucleic acid cargo as defined herein and apolymeric carrier molecule according to generic formula (I) or (Ia) oraccording to any of subformulas thereof as defined herein and optionallya pharmaceutically acceptable carrier and/or vehicle.

As a first ingredient, the inventive pharmaceutical compositioncomprises the polymeric carrier cargo complex formed by the nucleic acidcargo and a polymeric carrier molecule according to generic formula (I)or (Ia) or according to any of subformulas thereof as defined herein.

As a second ingredient the inventive pharmaceutical composition maycomprise at least one additional pharmaceutically active component. Apharmaceutically active component in this connection is a compound thathas a therapeutic effect to heal, ameliorate or prevent a particularindication, preferably cancer diseases, autoimmune disease, allergies orinfectious diseases. Such compounds include, without implying anylimitation, peptides or proteins, preferably as defined herein forcoding nucleic acids, nucleic acids, preferably as defined herein,(therapeutically active) low molecular weight organic or inorganiccompounds (molecular weight less than 5000, preferably less than 1000),sugars, antigens or antibodies, preferably as defined herein,therapeutic agents already known in the prior art, antigenic cells,antigenic cellular fragments, cellular fractions; cell wall components(e.g. polysaccharides), modified, attenuated or de-activated (e.g.chemically or by irradiation) pathogens (virus, bacteria etc.),adjuvants, preferably as defined herein, etc.

Furthermore, the inventive pharmaceutical composition may comprise apharmaceutically acceptable carrier and/or vehicle. In the context ofthe present invention, a pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of the inventive pharmaceuticalcomposition. If the inventive pharmaceutical composition is provided inliquid form, the carrier will typically be pyrogen-free water; isotonicsaline or buffered (aqueous) solutions, e.g. phosphate, citrate etc.buffered solutions. Particularly for injection of the inventivepharmaceutical composition, water or preferably a buffer, morepreferably an aqueous buffer, may be used, containing a sodium salt,preferably at least 50 mM of a sodium salt, a calcium salt, preferablyat least 0.01 mM of a calcium salt, and optionally a potassium salt,preferably at least 3 mM of a potassium salt. According to a preferredembodiment, the sodium, calcium and, optionally, potassium salts mayoccur in the form of their halogenides, e.g. chlorides, iodides, orbromides, in the form of their hydroxides, carbonates, hydrogencarbonates, or sulfates, etc. Without being limited thereto, examples ofsodium salts include e.g. NaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄,examples of the optional potassium salts include e.g. KCl, KI, KBr,K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts include e.g. CaCl₂,CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Furthermore, organic anions of theaforementioned cations may be contained in the buffer. According to amore preferred embodiment, the buffer suitable for injection purposes asdefined herein, may contain salts selected from sodium chloride (NaCl),calcium chloride (CaCl₂) and optionally potassium chloride (KCl),wherein further anions may be present additional to the chlorides. CaCl₂can also be replaced by another salt like KCl. Typically, the salts inthe injection buffer are present in a concentration of at least 50 mMsodium chloride (NaCl), at least 3 mM potassium chloride (KCl) and atleast 0.01 mM calcium chloride (CaCl₂). The injection buffer may behypertonic, isotonic or hypotonic with reference to the specificreference medium, i.e. the buffer may have a higher, identical or lowersalt content with reference to the specific reference medium, whereinpreferably such concentrations of the afore mentioned salts may be used,which do not lead to damage of cells due to osmosis or otherconcentration effects. Reference media are e.g. liquids occurring in “invivo” methods, such as blood, lymph, cytosolic liquids, or other bodyliquids, or e.g. liquids, which may be used as reference media in “invitro” methods, such as common buffers or liquids. Such common buffersor liquids are known to a skilled person. Ringer-Lactate solution isparticularly preferred as a liquid basis.

According to another aspect, the inventive pharmaceutical compositionmay comprise an adjuvant. In this context, an adjuvant may be understoodas any compound, which is suitable to initiate or increase an immuneresponse of the innate immune system, i.e. a non-specific immuneresponse. With other words, when administered, the inventivepharmaceutical composition typically elicits an innate immune responsedue to the adjuvant, optionally contained therein. Such an adjuvant maybe selected from any adjuvant known to a skilled person and suitable forthe present case, i.e. supporting the induction of an innate immuneresponse in a mammal.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulrnonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

The inventive pharmaceutical composition may be used for human and alsofor veterinary medical purposes, preferably for human medical purposes,as a pharmaceutical composition in general or as a vaccine.

According to a particular preferred aspect, the inventive pharmaceuticalcomposition (or the inventive polymeric carrier cargo complex) may beprovided or used as an immunostimulating agent. In this context, theinventive pharmaceutical composition is preferably as defined above.More preferably, the nucleic acid of the inventive polymeric carriercargo complex, preferably contained in the pharmaceutical composition,is typically an immunostimulatory nucleic acid as defined herein, e.g. aCpG-DNA or an immunostimulatory RNA (isRNA). Alternatively oradditionally, the nucleic acid of the inventive polymeric carrier cargocomplex, preferably contained in the pharmaceutical composition, is acoding nucleic acid as defined herein, preferably a cDNA or an mRNA,more preferably encoding an adjuvant protein preferably as definedherein.

In a specific aspect of this embodiment in this context it is preferredthat an adjuvant protein is a component of the polymeric carrier,preferably as (AA)_(x), component.

According to an even more preferred aspect, the inventive pharmaceuticalcomposition (or the inventive polymeric carrier cargo complex) may beprovided or used as an adjuvant. In this context, the adjuvant ispreferably defined as the inventive pharmaceutical composition above.More preferably, the nucleic acid of the inventive polymeric carriercargo complex, preferably contained in the adjuvant, is typically animmunostimulatory nucleic acid as defined herein, e.g. a CpG-DNA or animmunostimulatory RNA (isRNA). Alternatively or additionally, thenucleic acid of the inventive polymeric carrier cargo complex,preferably contained in the adjuvant, is a coding nucleic acid asdefined herein, preferably a cDNA or an mRNA, more preferably encodingan adjuvant protein, preferably as defined herein. The inventivepolymeric carrier cargo complex, preferably contained in the adjuvant,typically initiates an innate immune response in the patient to betreated. Such an adjuvant may be utilized in any accompanying therapy,with any known vaccine or any further (known) therapeutic agent,preferably prior to, concurrent with or subsequent to administration ofthe main therapy, prior to, concurrent with or subsequent toadministration of a further (known) vaccine or a (known) furthertherapeutic agent.

The inventive polymeric carrier cargo complex or the inventivepharmaceutical composition as defined herein provided or used as anadjuvant is preferably capable of triggering a non-antigen-specific,(innate) immune reaction (as provided by the innate immune system),preferably in an immunostimulating manner. An immune reaction cangenerally be brought about in various ways. An important factor for asuitable immune response is the stimulation of different T-cellsub-populations. T-lymphocytes typically differentiate into twosub-populations, the T-helper 1 (Th1) cells and the T-helper 2 (Th2)cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of effector proteins(cytokines) produced by them. Thus, Th1 cells assist the cellular immuneresponse by activation of macrophages and cytotoxic T-cells. Th2 cells,on the other hand, promote the humoral immune response by stimulation ofB-cells for conversion into plasma cells and by formation of antibodies(e.g. against antigens). The Th1/Th2 ratio is therefore of greatimportance in the immune response. In connection with the presentinvention, the Th1/Th2 ratio of the immune response is preferablydisplaced by the immune-stimulating agent, namely the inventivepolymeric carrier cargo complex in the direction towards the cellularresponse, that is to say the Th1 response, and a predominantly cellularimmune response is thereby induced. As defined above, the inventivepolymeric carrier cargo complex exerts by itself an unspecific innateimmune response, which allows the inventive polymeric carrier cargocomplex be used as such (without adding another pharmaceutically activecomponent) as an immunostimulating agent. If administered together withanother pharmaceutically active component, preferably a specificallyimmunogenic component, preferably an antigen, the nucleic acid of theinvention serves as an adjuvant supporting the specific adaptive immuneresponse elicited by the other pharmaceutically active component e.g. anantigen.

According to another particularly preferred embodiment, the inventivepharmaceutical composition (or the inventive polymeric carrier cargocomplex) may be provided or used as a vaccine.

Such an inventive vaccine is typically composed like the inventivepharmaceutical composition and preferably supports or elicits an immuneresponse of the immune system of a patient to be treated, e.g. an innateimmune response, if an RNA or mRNA is used as the nucleic acid moleculeof the inventive polymeric carrier cargo complex formed by the nucleicacid cargo and a polymeric carrier molecule according to generic formula(I) or (Ia) or according to any of subformulas thereof as definedherein. Furthermore or alternatively, the inventive vaccine may elicitan adaptive immune response, preferably, if the nucleic acid of theinventive polymeric carrier cargo complex formed by the nucleic acidcargo and a polymeric carrier molecule according to generic formula (I)or (Ia) or according to any of subformulas thereof as defined hereinencodes any of the above mentioned antigens or proteins, which elicit anadaptive immune response.

In this context, the vaccine is preferably defined as an adjuvant or asan inventive pharmaceutical composition as disclosed above. Morepreferably, the nucleic acid of the inventive polymeric carrier cargocomplex, contained in such a vaccine, may be any nucleic acid as definedabove, preferably an immunostimulatory nucleic acid as defined herein,e.g. a CpG-DNA or an immunostimulatory RNA (isRNA). Alternatively oradditionally, the nucleic acid of the inventive polymeric carrier cargocomplex, preferably contained in the vaccine, is a coding nucleic acidas defined herein, preferably a cDNA or an mRNA, more preferablyencoding an adjuvant protein, preferably as defined herein.Alternatively or additionally, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in the vaccine, isa coding nucleic acid as defined herein, preferably a cDNA or an mRNA,more preferably encoding an antigen, preferably as defined herein.Furthermore, particularly, if the nucleic acid of the inventivepolymeric carrier cargo complex does not encode an antigen, theinventive vaccine may contain an antigen, preferably as defined above,either as a protein or peptide or encoded by a nucleic acid, orantigenic cells, antigenic cellular fragments, cellular fractions; cellwall components (e.g. polysaccharides), modified, attenuated orde-activated (e.g. chemically or by irradiation) pathogens (virus,bacteria etc.).

According to a further aspect the inventive vaccine may contain apeptide or protein antigen as (AA)_(x) component of the inventivepolymeric carrier as defined herein, preferably as part of therepetitive component [S—P²—S]_(n).

The inventive vaccine may also comprise a pharmaceutically acceptablecarrier, adjuvant, and/or vehicle as defined herein for the inventivepharmaceutical composition.

The inventive vaccine can additionally contain one or more auxiliarysubstances in order to increase its immunogenicity, if desired. Asynergistic action of the inventive polymeric carrier cargo complexformed by the nucleic acid cargo and a polymeric carrier moleculeaccording to generic formula (I) or (Ia) or according to any ofsubformulas thereof as defined herein and of an auxiliary substance,which may be optionally contained in the inventive vaccine as definedherein, is preferably achieved thereby. Depending on the various typesof auxiliary substances, various mechanisms can come into considerationin this respect. For example, compounds that permit the maturation ofdendritic cells (DCs), for example lipopolysaccharides, TNF-alpha orCD40 ligand, form a first class of suitable auxiliary substances. Ingeneral, it is possible to use as auxiliary substance any agent thatinfluences the immune system in the manner of a “danger signal” (LPS,GP96, etc.) or cytokines, such as GM-CFS, which allow an immune responseto be enhanced and/or influenced in a targeted manner. Particularlypreferred auxiliary substances are cytokines, such as monokines,lymphokines, interleukins or chemokines, that further promote the innateimmune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF,G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

Further additives which may be included in the inventive vaccine areemulsifiers, such as, for example, Tween®; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

The inventive vaccine can also additionally or alternatively contain anyfurther compound, which is known to be immune-stimulating due to itsbinding affinity (as ligands) to human Toll-like receptors TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventive vaccinein this context, may be CpG nucleic acids, in particular CpG-RNA orCpG-DNA. A CpG-RNA or CpG-DNA can be a single-stranded CpG-DNA (ssCpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA(ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleicacid is preferably in the form of CpG-RNA, more preferably in the formof single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid preferablycontains at least one or more (mitogenic) cytosine/guanine dinucleotidesequence(s) (CpG motif(s)). According to a first preferred alternative,at least one CpG motif contained in these sequences, that is to say theC (cytosine) and the G (guanine) of the CpG motif, is unmethylated. Allfurther cytosines or guanines optionally contained in these sequencescan be either methylated or unmethylated. According to a furtherpreferred alternative, however, the C (cytosine) and the G (guanine) ofthe CpG motif can also be present in methylated form.

The inventive vaccine can also additionally or alternatively contain animmunostimulatory RNA, i.e. an RNA derived from an immunostimulatoryRNA, which triggers or increases an (innate) immune response.Preferably, such an RNA may be in general as defined herein for RNAs. Inthis context, those classes of RNA molecules, which can induce an innateimmune response, may be selected e.g. from ligands of Toll-likereceptors (TLRs), particularly from RNA sequences representing and/orencoding ligands of TLRs, preferably selected from human family membersTLR1-TLR10 or murine family members TLR1-TLR13, more preferably fromTLR7 and TLR8, ligands for intracellular receptors for RNA (such asRIG-I or MDA-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006).Toll-like receptors and RNA helicases: two parallel ways to triggerantiviral responses. Mol. Cell 22, 561-569), or any otherimmunostimulatory RNA sequence. Such an immunostimulatory RNA maycomprise a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30nucleotides.

The present invention furthermore provides several applications and usesof the inventive polymeric carrier molecule according to generic formula(I) or (Ia) or according to any of subformulas thereof as definedherein, the inventive polymeric carrier cargo complex formed by thenucleic acid cargo and the inventive polymeric carrier molecule, apharmaceutical composition comprising same or of kits comprising same.

According to one embodiment, the present invention is directed to thefirst medical use of the inventive polymeric carrier molecule accordingto generic formula (I) or (Ia) or according to any of subformulasthereof as defined herein, of the inventive polymeric carrier cargocomplex formed by the nucleic acid cargo and the inventive polymericcarrier molecule, or of kits comprising same, as a medicament,preferably for gene therapy or treatment of a disease as defined herein.The medicament may be in the form of a pharmaceutical composition or inthe form of an adjuvant or a vaccine as a specific form ofpharmaceutical compositions. A pharmaceutical composition in the contextof the present invention typically comprises the inventive polymericcarrier molecule according to generic formula (I) or (Ia) or accordingto any of subformulas thereof as defined herein or the inventivepolymeric carrier cargo complex formed by the nucleic acid cargo and theinventive polymeric carrier molecule, optionally further ingredients,e.g. as defined herein for the inventive nucleic acid, and optionally apharmaceutically acceptable carrier and/or vehicle, preferably asdefined herein.

According to one further embodiment, the present invention is directedto the use of the inventive polymeric carrier molecule according togeneric formula (I) or (Ia) or according to any of subformulas thereofas defined herein, preferably with a nuleic acid cargo as definedherein, or of the inventive polymeric carrier cargo complex formed bythe nucleic acid cargo and the inventive polymeric carrier molecule forthe prophylaxis, treatment and/or amelioration of diseases as definedherein. Preferably, diseases as mentioned herein are selected fromcancer or tumour diseases, infectious diseases, preferably (viral,bacterial or protozoological) infectious diseases, autoimmune diseases,allergies or allergic diseases, monogenetic diseases, i.e. (hereditary)diseases, or genetic diseases in general, diseases which have a geneticinherited background and which are typically caused by a defined genedefect and are inherited according to Mendel's laws, cardiovasculardiseases, neuronal diseases, diseases of the respiratory system,diseases of the digestive system, diseases of the skin, musculoskeletaldisorders, disorders of the connective tissue, neoplasms, immunedeficiencies, endocrine, nutritional and metabolic diseases, eyediseases, ear diseases and any disease which can be influenced by thepresent invention.

According to another embodiment, the present invention is directed tothe second medical use of the inventive polymeric carrier moleculeaccording to generic formula (I) or (Ia) or according to any ofsubformulas thereof as defined herein, or of the inventive polymericcarrier cargo complex formed by the nucleic acid cargo and the inventivepolymeric carrier molecule for the treatment of diseases as definedherein, preferably to the use of the inventive polymeric carriermolecule according to generic formula (I) or (Ia) or according to any ofsubformulas thereof as defined herein, or of the inventive polymericcarrier cargo complex formed by the nucleic acid cargo and the inventivepolymeric carrier molecule, of a pharmaceutical composition comprisingsame or of kits comprising same for the preparation of a medicament forthe prophylaxis, treatment and/or amelioration of various diseases asdefined herein.

According to one further embodiment, the present invention is directedto the use of the inventive polymeric carrier molecule according togeneric formula (I) or (Ia) or according to any of subformulas thereofas defined herein, preferably with a nucleic acid cargo as definedherein, or of the inventive polymeric carrier cargo complex formed bythe nucleic acid cargo and the inventive polymeric carrier molecule forimmunotherapy, for gene therapy, for vaccination, or to the use thereofas an adjuvant.

According to a further embodiment, the present invention is directed tothe treatment of diseases as defined herein, particularly prophylaxis,treatment and/or amelioration of various diseases as defined herein,preferably using or administering to a patient in need thereof theinventive polymeric carrier cargo complex formed by the nucleic acidcargo and the inventive polymeric carrier molecule according to genericformula (I) or (Ia) or according to any of subformulas thereof asdefined herein, or the inventive pharmaceutical composition or vaccineas defined herein.

The present invention also allows treatment of diseases, which have notbeen inherited, or which may not be summarized under the abovecategories. Such diseases may include e.g. the treatment of patients,which are in need of a specific protein factor, e.g. a specifictherapeutically active protein as mentioned above. This may e.g. includedialysis patients, e.g. patients which undergo a (regular) a kidney orrenal dialysis, and which may be in need of specific therapeuticallyactive proteins as defined herein, e.g. erythropoietin (EPO), etc.

According to a final embodiment, the present invention also provideskits, particularly kits of parts, comprising as components alone or incombination with further ingredients at least one inventive polymericcarrier molecule according to generic formula (I) or (Ia) or accordingto any of subformulas thereof as defined herein, at least one inventivepolymeric carrier cargo complex formed by the nucleic acid cargo and theinventive polymeric carrier molecule, at least one nucleic acid asdefined herein, at least one pharmaceutical composition comprising sameand/or kits comprising same, and optionally technical instructions withinformation on the administration and dosage of the inventive polymericcarrier molecule, the nucleic acid, the inventive polymeric carriercomplex, and/or the inventive pharmaceutical composition. Such kits,preferably kits of parts, may be applied, e.g., for any of the abovementioned applications or uses. Such kits, when occurring as a kit ofparts, may further contain each component in a different part of thekit.

FIGURES

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1: illustrates the products formed during the reaction by SDS PAGEgel electrophoresis under non-reducing conditions. The left part of thegel was stained according to coomassie protocol which colors the peptidecontent, the right part was stained with Bariumchloride/Iodine solutionswhich colour the PEG content. As may be easily seen only one productcontaining PEG and peptide is formed in the range of the intended mass.

FIG. 2: shows the results of the confocal microscopy of L929 cells 5minutes after transfection with fluorescence labelled RNA complexed withthe inventive polymeric carrier PB19 in a molar ratio of 1:500. As aresult, several complexes are detectable in the cells already 5 minutesafter transfection indicating a good transfection rate.

FIG. 3: shows the results of the confocal microscopy of L929 cells 1hour after transfection with fluorescence labelled RNA complexed withthe inventive polymeric carrier PB19 in a molar ratio of 1:500. As aresult, most of the particles were taken up in the cells 1 hour aftertransfection showing a good transfection rate.

FIG. 4: illustrates stability experiments with regard to electrostaticdisplacement of the bound nucleic acid from the complex. As can be seenthe addition of the anionic polymer heparin can not displace the RNAfrom the complex because it still migrates in the gel. This indicatesthat the complex binding is so strong that a competitive complex partnercannot displace the RNA from the complex.

FIG. 5: depicts a gel shift assay to examine the strength of complexbinding. It could be shown that the addition of the anionic polymerheparin or the reducing agent DTT alone cannot display the RNA from thecomplex with the polymer according to the invention (PB19). Onlytogether they are able to displace the RNA from the complex (lane 7).This indicates that the complex binding is so strong that neither acompetitive complex partner nor a reducing agent can displace the RNAfrom the complex.

FIG. 6: shows a gel shift assay to examine the strength of complexbinding. As can be seen, the addition of the anionic polymer heparinalone cannot displace the RNA from the complex with polymers accordingto the invention (PB22). In contrast mRNA could be readily displaced byheparin from PEI complexes.

FIG. 7: depicts the results from expression experiments with ofluciferase encoding mRNA according to SEQ ID NO: 369 in HeLa cells. Ascan be seen, formulations of mRNA coding for luciferase (luc-RNActive)according to SEQ ID NO: 369 with the PB19 polymer (molar ratio ofRNA:PB19 1:1000, 1:500, 1:100) lead to expression of luciferaseindependently of the presence of serum containing medium. These resultsare unexpected because serum containing medium leads in general to aloss of transfection efficiency.

FIG. 8: depicts the results from expression experiments with ofluciferase encoding mRNA according to SEQ ID NO: 369 in HeLa cells. Ascan be seen, formulations of mRNA coding for luciferase (luc-RNActive)according to SEQ ID NO: 369 with the PB83 polymer (molar ratio ofRNA:PB83 1:50) lead to significant higher expression of luciferasecompared to th free peptide combined with RNA in molar ratio 1:50 (e.g.template assisted polymerization in situ Rice et. Al.), to thenon-pegylated polymerization product PB83 w/o PEG in molar ratio 1:50(e.g. RPC conform), PB83 polymerization without peptide component (e.g.dimerized PEG-SS-PEG) and a non-reversible PEGylated of PB83 derivativewhich was synthesized by malimide containing PEG termination of thepolymerization reaction.

FIG. 9A: illustrates the results from expression experiments withluciferase encoding mRNA according to SEQ ID NO: 369 in BALB/c miceafter intradermal injection. As a result, formulation of mRNA coding forluciferase (luc-RNActive) according to SEQ ID NO: 369 with the PB19polymer leads to expression of luciferase in the dermis of female BALB/cmice. Other transfection reagents known in the art (PEI andLipofectamine 2000) did not show any expression of the Luciferaseprotein.

FIG. 9B: illustrates the results from expression experiments withluciferase encoding mRNA according to SEQ ID NO: 369 in BALB/c miceafter intramuscular injection. As a result, formulation of mRNA codingfor luciferase (luc-RNActive) according to SEQ ID NO: 369 with the PB19polymer leads to expression of luciferase in the m. tibialis of femaleBALB/c mice. Other transfection reagents known in the art (PEI andLipofectamine 2000) did not show any expression of the Luciferaseprotein.

FIG. 10: shows the expression of luciferase in BALB/c mice afterintradermal injection of different formulations of mRNA coding forluciferase (luc-RNActive) according to SEQ ID NO: 369 with two differentpolymers according to the invention [PB19 and PB48]. These formulationsof mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 369with two different polymers according to the invention [PB19 and PB48]lead to expression of luciferase in the dermis of female BALB/c mice.The polymeric carrier cargo complex formed by a peptide according to RPCCH6R4H6C (without PEGylation) and mRNA coding for luciferase(luc-RNActive) according to SEQ ID NO: 369 in a molar ratio of 2500:1showed no expression of luciferase after intradermal injection of thecomplexed mRNA.

FIG. 11: shows the expression of luciferase in BALB/c mice afterintradermal injection of different formulations of mRNA coding forluciferase (luc-RNActive) according to SEQ ID NO: 369 with threedifferent polymers according to the invention [PB124, PB117 and PB83].These formulations lead all to an expression of luciferase in the dermisof female BALB/c mice.

FIG. 12: depicts the results from expression experiments with ofluciferase encoding mRNA according to SEQ ID NO: 369 in CHO cells. Ascan be seen, formulations of mRNA coding for luciferase (luc-RNActive)according to SEQ ID NO: 369 with different polymers according to theinvention (molar ratio of RNA:PB 1:250) all lead to high levels ofluciferase expression in serum containing medium. Also the advantageouseffect of the hydrophilic component AA (in this case the peptideCAS₃PS₃AC in the polymer can easily be seen.

FIG. 13: depicts the secretion of hIL-6 cytokine in hPBMCs. It could beshown that complexes consisting of the polymers according to theinvention (PB19 and PB22) and mRNA coding for luciferase (luc-RNActive)according to SEQ ID NO: 369 do not induce the secretion of cytokines inhPBMCs in contrast to complexes consisting of mRNA coding for luciferase(luc-RNActive) according to SEQ ID NO: 369 and cationic peptides(H3R9H3) or a combination of cationic peptides (H3R9H3) and PEGylatedcationic peptides (which confers in general to subsequent hydrophiliccoating of pre-formed nucleic acid condensates). The polymers used were:

-   -   E9-PEG5k: HO-PEG₅₀₀₀-EEEEEEEEE    -   E9-PEG3k: HO-PEG₃₀₀₀-EEEEEEEEE    -   R9-PEG3k: HO-PEG₃₀₀₀-RRRRRRRRR    -   PB19: HO-PEG₅₀₀₀-S—(S—CHHHRRRRHHHC—S)₅—S-PEG₅₀₀₀-OH    -   PB22: HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S)₅—S-PEG₅₀₀₀-OH

FIG. 14: illustrates the secretion of hTNFa cytokine in hPBMCs. It couldbe shown that complexes consisting of the polymers according to theinvention (PB19 and PB22) and mRNA coding for luciferase (luc-RNActive)according to SEQ ID NO: 369 do not induce the secretion of cytokines inhPBMCs in contrast to complexes consisting of mRNA coding for luciferase(luc-RNActive) according to SEQ ID NO: 369 and cationic peptides(H3R9H3) or such complexes coated with PEGylated peptides (which confersin general to subsequent hydrophilic coating of pre-formed nucleic acidcondensates). The polymers used were:

-   -   E9-PEG5k: HO-PEG₅₀₀₀-EEEEEEEEE    -   E9-PEG3k: HO-PEG₃₀₀₀-EEEEEEEEE    -   R9-PEG3k: HO-PEG₃₀₀₀-RRRRRRRRR    -   PB19: HO-PEG₅₀₀₀-S—(S—CHHHRRRRHHHC—S)₅—S-PEG₅₀₀₀-OH    -   PB22: HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC-5)₅-S—PEG₅₀₀₀-OH

FIG. 15: shows the secretion of hTFNa cytokine secretion in hPBMCs. Itcould be shown that complexes consisting of mRNA coding for luciferase(luc-RNActive) according to SEQ ID NO: 369 and polymers according to theinvention (PB19) do not induce the secretion of hTFNa in hPBMCs incontrast to complexes consisting of mRNA coding for luciferase(luc-RNActive) according to SEQ ID NO: 369 and state of the arttransfection reagents like Lipofectamin 2000.

FIG. 16: shows the mRNA sequence encoding Photinus pyralis luciferase(SEQ ID NO: 369) in the mRNA construct pCV19-Pp luc(GC)-muag-A70-C30;which exhibits a length of 1857 nucleotides. The mRNA sequence containsfollowing sequence elements:

-   -   the coding sequence encoding Photinus pyralls luciferase;    -   stabilizing sequences derived from alpha-globin-3′-UTR (muag        (mutated alpha-globin-3′-UTR));    -   70× adenosine at the 3′-terminal end (poly-A-tail);    -   30× cytosine at the 3′-terminal end (poly-C-tail).    -   The ORF is indicated in italic letters, muag (mutated        alpha-globin-3′-UTR is indicated with a dotted line, the        poly-A-tail is underlined with a single line

EXAMPLES

The following examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

1. Preparation of DNA and mRNA Constructs Encoding Pp Luciferase(Photinus pyralis)

For the present examples DNA sequences, encoding Photinus pyralisluciferase, were prepared and used for subsequent in vitro transcriptionreactions.

According to a first preparation, the DNA sequence termedpCV19-Ppluc(GC)-muag-A70-C30 sequence was prepared, which corresponds tothe Photinus pyralis luciferase coding sequence. The construct wasprepared by modifying the wildtype Photinus pyralis luciferase encodingDNA sequence by introducing a GC-optimized sequence for a better codonusage and stabilization, stabilizing sequences derived fromalpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)), a stretch of70× adenosine at the 3′-terminal end (poly-A-tail) and a stretch of 30×cytosine at the 3′-terminal end (poly-C-tail), leading to SEQ ID NO: 369(see FIG. 16). The sequence of the final DNA construct had a length of1857 nucleotides and was termed “pCV19-Ppluc(GC)-muag-A70-C30”. In SEQID NO: 369 (see FIG. 16) the sequence of the corresponding mRNA isshown.

The sequence contains following sequence elements:

-   -   the coding sequence encoding Photinus pyralis luciferase;    -   stabilizing sequences derived from alpha-globin-3′-UTR (muag        (mutated alpha-globi n-3′-UTR));    -   70× adenosine at the 3′-terminal end (poly-A-tail);    -   30× cytosine at the 3′-terminal end (poly-C-tail).

2. In Vitro Transcription:

The respective DNA plasmid prepared according to Example 1 wastranscribed in vitro using T7-Polymerase. Subsequently the mRNA waspurified using PureMessenger® (CureVac, Tubingen, Germany).

3. Reagents:

Peptides: The peptides used in the present experiments were as follows:

-   -   PB19: HO-PEG₅₀₀₀-S—(S—CHHHRRRRHHHC—S)₅—S-PEG₅₀₀₀-OH (pegylated        CH₃R₄H₃C peptide polymer)    -   PB22: HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S)₅—S-PEG₅₀₀₀-OH        (pegylated CH₆R₄H₆C peptide polymer)    -   PB48: HO-PEG₅₀₀₀-S—(S—CHHHRRRRHHHC—S)₃—S-PEG₅₀₀₀-OH    -   PB83: HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S-)₇-S-PEG₅₀₀₀-OH    -   PB86:        HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S-)₅(S-CAS3PS3AC—S)₅—S-PEG₅₀₀₀-OH    -   PB117 HO-PEG₅₀₀₀-S—(S—CHKKKKKKHC—S-)₇-S-PEG₅₀₀₀-OH    -   PB124 HO-PEG₂₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S-)₇-S-PEG₂₀₀₀-OH    -   PB83 free peptide HS—(CHHHHHHRRRRHHHHHHC)—SH freshly solved in        water prior formulation    -   PB83 w/o peptide PEG-SS-PEG    -   PB83 w/o PEG HS—(CHHHHHHRRRRHHHHHHC)—SH    -   PB83malPEG PEG-mal-(S—CHHHHHHRRRRHHHHHHC—S-)_(x)-mal-PEG    -   H3R9H3: HHHRRRRRRRRRHHH    -   CH6R4H6C: H—(S—CHHHHHHRRRRHHHHHHC—S)₅—H

Further Tranfection Reagents Used are:

-   -   Lipofectamine 2000 (Invitrogen)    -   PEI 25 kDa (branched) (Aldrich)

4. Synthesis of the Inventive Polymeric Carrier:

The condensation reaction was performed with the calculated amount ofpeptide (component P²) which is dissolved in a mixture of a bufferedaqueous solution at pH 8.5 with an optional additive of 5% (v/v)Dimethylsulfoxide (DMSO) (which are mild oxidation conditions andtherefore allow the establishment of an equilibrium) and stirred for 18h at ambient temperature. Afterwards the calculated amount of a thiolgroup containing PEG derivative (alpha-Methoxy-omega-mercaptopoly(ethylene glycol)) (component P¹) (dissolved in water) is added andthe resulting solution is stirred for another 18 h. Subsequentlyophilisation and purification yield the desired polymer. The ratiobetween PEG component P¹ to peptide component P² defines the chainlength of the P² polymer.

The condensation reaction in this reaction environment is reversible,therefore the chain length of the polymer is determined by the amount ofthe monothiol compound which terminates the polymerisation reaction. Insummary the length of the polymer chain is determined by the ratio ofoligo-peptide and monothiol component. This reaction is supported by thechosen mild oxidation conditions. With more stringent oxidationconditions (30% DMSO) the generation of high molecular (long chain)polymers is induced.

4.1. 1. Step: Exemplary Polymerization Reaction:

nHS—CHHHRRRHHHC—SH→H—(S—CHHHRRRRHHHC—S)_(n)—H

4.2. 2. Step: Exemplary Stop Reaction:

H—(S—CHHHRRRRHHHC—S)_(n)H+2PEG-SH→PEG-S—(S—CHHHRRRRHHHC—S)_(n)—S-PEG

4.3. Exemplary Synthesis Reaction:

-   -   step 1)

5×HS—CHHHRRRHHHC—SH→H—(S—CHHHRRRRHHHC—S)₅—H

-   -   step 2)

H—(S—CHHHRRRRHHHC—S)₅—H+2×PEG₅₀₀₀-SH-PEG₅₀₀₀-S—(S—CHHHRRRRHHHC—S)₅—S-PEG₅₀₀₀

To achieve a polymer length of 5 (n=5), a molar ratio of peptide:PEG of5:2 was used.

Some variations of the synthesis reaction were done to show the theeffect of the PEGchains and the effects of the reversible attachment ofthe PEG chains.

4.4. Synthesis Reaction for Polymeric Carriers without PEG Chains:

The reaction conditions are the same as mentioned above, but the step ofthe addition of a sulfhydryl containing PEG derivative is notperformed/skipped.

4.5. Synthesis Reaction for Irreversible Attached PEG Chains:

The reaction conditions are the same as mentioned above, but instead ofa sulfhydryl containing PEG derivative a maleimide containing PEGderivative is utilized. The maleimide mojety reacts rapidly with freesulfhydryl groups forming a covalent bond. Therefore the termination ofthe polymerization is not under the dynamic equilibria conditions as forsulfhyrdyl containing PEG derivatives but under irreversible conditionswhich results in a “frozen” polymerization pattern of high polydiversityand not the defined reaction products of the dynamic equilibriareaction.

5. Complexation of RNA:

The mRNA construct defined above in Example 1 and prepared according toExample 2, were complexed for the purposes of the present invention withthe polymers, preferably as defined in Example 4. Therefore, 4 μg RNAcoding for luciferase pCV19-Ppluc(GC)-muag-A70-C30 (Luc-RNActive)according to SEQ ID NO: 85 were mixed in molar ratios as indicated withthe inventive polymeric carrier (according to formula I) or a control,thereby forming a complex. Afterwards the resulting solution wasadjusted with water to a final volume of 50 μl and incubated for 30minutes at room temperature.

The different inventive polymeric carriers and the different ratios ofpolymeric carriers/RNA used in this experiment are shown in table 1.

Cationic Präfix Polymer Ratio AS N/P PB19HO-PEG₅₀₀₀-S-(S-CHHHRRRRHHHC-S)₅-S-PEG₅₀₀₀-OH 500 20 5.6 PB19HO-PEG₅₀₀₀-S-(S-CHHHRRRRHHHC-S)₅-S-PEG₅₀₀₀-OH 250 20 2.8 PB19HO-PEG₅₀₀₀-S-(S-CHHHRRRRHHHC-S)₅-S-PEG₅₀₀₀-OH  50 20 0.6 PB48HO-PEG₅₀₀₀-S-(S-CHHHRRRRHHHC-S)₃-S-PEG₅₀₀₀-OH 250 12 1.67 PB76HO-PEG₅₀₀₀-S-(S-CHHHHHHRRRRHHHHHHC-S)₁₀-S-PEG₅₀₀₀-OH 500 40 11.2 PB76HO-PEG₅₀₀₀-S-(S-CHHHHHHRRRRHHHHHHC-S)₁₀-S-PEG₅₀₀₀-OH 250 40 5.6 PB76HO-PEG₅₀₀₀-S-(S-CHHHHHHRRRRHHHHHHC-S)₁₀-S-PEG₅₀₀₀-OH  50 40 1.1 PB83HO-PEG₅₀₀₀-S-(S-CHHHHHHRRRRHHHHHHC-S-)₇-S-PEG₅₀₀₀-OH 250 28 2.8 PB86HO-PEG₅₀₀₀-S-(S-CHHHHHHRRRRHHHHHHC-S-)₅(S-CAS3PS3AC-S)₅- 250 20 2.8S-PEG₅₀₀₀-OH Ratio = molar ratio of RNA:peptide cationic AS = cationicamino acids, which are positively charged at a physiological pH (i.e.not histidine (H) but e.g. arginine (R)) Whereas PB83 and PB86 have acationic insert that is double in size compared to PB19, therefore hasdouble as much cationic amino acid residues. PB48 has a shorter cationicinsert compared to PB19, therefore has less cationic amino acid residuesper molecule polymer. N/P = is the ratio of basic nitrogen atoms in thepolymeric carrier to phosphate residues in the nucleic acid, consideringthat nitrogen atoms confer to positive charges and phosphate of thephosphate backbone of the nucleic acid confers to the negative charge.Histidine residues are counted neutral, because complex formation isdone at physiological pH, therefore the imidazole residue is uncharged.N/P is calculated by the following formula:${N\text{/}P} = \frac{{{pmol}\mspace{14mu}\lbrack{RNA}\rbrack}*{ratio}*{cationic}\mspace{14mu} {AS}}{{\mu g}\mspace{14mu} {RNA}*3*1000}$For the calculations RNA coding for luciferasepCV19-Ppluc(GC)-muag-A70-C30 (Luc-RNActive) according to SEQ ID NO: 369was applied, which has a molecular weight of 660 kDa. Therefore 1 μgpCV19-Ppluc(GC)-muag-A70-C30 RNA according to SEQ ID NO: 369 confers to1.67 pmol pCV19-Ppluc(GC)-muag-A70-C30 RNA according to SEQ ID NO: 369

6. Size and Zetapotential Measurements:

The hydrodynamic diameters of polyplexes as prepared above were measuredby dynamic light scattering using a Zetasizer Nano (Malvern Instruments,Malvern, UK) according to the SOPs distributed by Malvern. Themeasurements were performed at 25° C. in the specified buffer analysedby a cumulant method to obtain the hydrodynamic diameters andpolydispersity indices of the polyplexes.

The Zeta potential of the polyplexes was evaluated by the laser Dopplerelectrophoresis method using a Zetasizer Nano (Malvern Instruments,Malvern, UK). The measurement was performed at 25° C. and a scatteringangle of 173° was used.

7. Gel Shift Assay

Furthermore, mRNA coding for luciferase (Luc-RNActive) according to SEQID NO: 369 was formulated with the polymers as indicated and aliquotswere incubated with either heparin (100 μg) or Dithiothreitol (DTT) for15 Minutes at 37° C. Afterwards electrophoresis was done on agarose geland the nucleic acids were visualized by ethidium bromide staining.

8. Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy was performed on an inverted LSM510laser scanning microscope (Carl Zeiss, Germany) using a Plan-Apochromat63×/1.4 N.A. lens. All analyses were performed with living, nonfixedcells grown in eight-well chambered cover slides (Nunc, Germany). Forthe detection of Alexa Fluor 647 labelled mRNA only the light of a633-nm helium neon laser, directed over a UV/488/543/633 beam splitterin combination with a LP 650 long pass filter was used. Life cellmicroscopy was performed at room temperature.

For this purpose, L929 cells (25×10³/well) were seeded 1 day prior totransfection on 24-well microtiter plates leading to a 70% confluencewhen transfection was carried out. Cells were transfected withformulations containing 2 μg Alexa Fluor 647 labelled mRNA in 8 chamberwell slides directly before conduction of the microscopy experiment.

8. Cytokinstimulation in hPBMC

HPBMC cells from peripheral blood of healthy donors were isolated usinga Ficoll gradient and washed subsequently with 1×PBS (phophate-bufferedsaline). The cells were then seeded on 96-well microtiter plates(200×10³/well). The hPBMC cells were incubated for 24 h with 10 μl ofthe RNA/carrier complex in X-VIVO 15 Medium (BioWhittaker). As RNA SEQID NO: 369 was used. The carriers were as shown above for genericformula (I). The immunostimulatory effect upon the hPBMC cells wasmeasured by detecting the cytokine production (Interleukin-6; Tumornecrose factor alpha, Interferon alpha). Therefore, ELISA microtiterplates (Nunc Maxisorb) were incubated over night (o/n) with bindingbuffer (0.02% NaN₃, 15 mM Na₂CO₃, 15 mM NaHCO₃, pH 9.7), additionallycontaining a specific cytokine antibody. Cells were then blocked with1×PBS, containing 1% BSA (bovine serum albumin). The cell supernatantwas added and incubated for 4 h at 37° C. Subsequently, the microtiterplate was washed with 1×PBS, containing 0.05% Tween-20 and thenincubated with a Biotin-labelled secondary antibody (BD Pharmingen,Heidelberg, Germany). Streptavidin-coupled horseraddish peroxidase wasadded to the plate. Then, the plate was again washed with 1×PBS,containing 0.05% Tween-20 and ABTS(2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added as asubstrate. The amount of cytokine was determined by measuring theabsorption at 405 nm (OD 405) using a standard curve with recombinantcytokines (BD Pharmingen, Heidelberg, Germany) with the SunriseELISA-Reader from Tecan (Crailsheim, Germany).

9. Transfection of HeLa Cells:

4 μg RNA stabilized luciferase mRNA (Luc-RNActive) according to SEQ IDNO: 85 were mixed in molar ratios as indicated with the respectivepolymer (according to formula I), thereby forming a complex. Afterwardsthe resulting solution was adjusted with water to a final volume of 50μl and incubated for 30 minutes at room temperature. The used ratios areindicated in table 1 above.

Hela-cells (150×10³/well) were seeded 1 day prior to transfection on24-well microtiter plates leading to a 70% confluence when transfectionwas carried out. For transfection 50 μl of the RNA/carrier complexsolution were mixed with 250 μl serum free or FCS containing medium (asindicated in the provided table) and added to the cells (final RNAconcentration: 13 μg/ml). Prior to addition of the serum freetransfection solution the HeLa-cells were washed gently and carefully 2times with 1 ml Optimen (Invitrogen) per well. Then, the transfectionsolution (300 μl per well) was added to the cells and the cells wereincubated for 4 h at 37° C. Subsequently 300 μl RPMI-medium (Camprex)containing 10% FCS was added per well and the cells were incubated foradditional 20 h at 37° C. The transfection solution was sucked off 24 hafter transfection and the cells were lysed in 300 μl lysis buffer (25mM Tris-PO₄, 2 mM EDTA, 10% glycerol, 1% Triton-X 100, 2 mM DTT). Thesupernatants were then mixed with luciferin buffer (25 mM Glycylglycin,15 mM MgSO₄, 5 mM ATP, 62.5 μM luciferin) and luminiscence was detectedusing a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad,Germany)).

10. Expression of Luciferase In Vivo:

10 μg mRNA coding for luciferase (Luc-RNActive) according to SEQ ID NO:369 were mixed in a molar ratio of 1:250 (RNA:polymeric carrier) withthe respective carrier (according to formula I), thereby forming acomplex. Afterwards the resulting solution was adjusted with RingerLactate solution to a final volume of 100 μl and incubated for 30minutes at room temperature, yielding a solution with a 0.1 g/lconcentration of complexed RNA.

100 μl (20 μl) of this solution was administrated intradermally (earpinna or back) or intramuscularly (m. tibialis) to 7 week old BALB/cmice. After 24 h the mice were sacrificed and the samples (ear, skinfrom the back or muscle) were collected, frozen at −78° C. and lysed for3 Minutes at full speed in a tissue lyser (Qiagen, Hilden, Germany).Afterwards 600 μl of lysis buffer were added and the resulting solutionswere subjected another 6 minutes at full speed in the tissue lyser.After 10 minutes centrifugation at 13500 rpm at 4° C. the supernatantswere mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO4, 5 mMATP, 62.5 μM luciferin) and luminiscence was detected using aluminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad,Germany)).

11. Results: 11.1. DLS/Zetasizer Determinations:

The size and ζ-potential of the polymeric carrier cargo complexesaccording to the invention were evaluated in triplicates by dynamiclight scattering (DLS) and laser-doppler electrophoresis and compared todifferent polymeric carrier cargo complexes known in the state of theart. Table 1 summarizes the cumulant diameters and the ζ-potential ofthe polymeric carrier cargo complexes.

Polymeric Cumulant Cumulant carrier diameter diameter ζ- cargo in waterin ringer potential complex N/P (nm) lactate (nm) (mV) PB19 6 58 ± 2  81± 3 −9 mV PEI 10 88 ± 4 110 ± 2 +20 mV RPC like 5 170 ± 8  >1000 +39 mVpolymer CH6R4H6C

The cumulant diameters of the polymeric carrier cargo complexes formedby polymers according to the invention and mRNA coding for luciferase(Luc-RNActive) according to SEQ ID NO: 369 showed that small, uniformcomplexes were formed. These complexes are stable against agglomerationin salt containing buffer and are less than 100 nm in size. In contrastpolymeric carrier cargo complexes according to the RPC procedure areunstable in salt containing buffers, forming large aggregates. Thepolymeric carrier cargo complexes according to the invention also formcomplexes of low ζ-potential, which is linked to a low tendency inbinding components of the serum, and therefore have a low tendency foropsonisation.

11.2. Confocal Microscopy:

The transfection of L929 cells with AlexaFlour647 aminoallyl-labelledRNA complexed with the inventive polymer PB19 after 5 minutes alreadyled to detectable complexes in the cell. The results are shown in FIGS.2 and 3. As can be seen in FIG. 2, confocal microscopy of L929 cells 5minutes after transfection with fluorescence labelled RNA (SEQ ID NO:369) complexed with the inventive polymer PB19 in a molar ratio of 1:500showed that already 5 minutes after transfection several complexes aredetectable in the cells. After 1 h confocal microscopy of L929 cells 1 hafter transfection with fluorescence labelled RNA complexed with theinventive polymer PB19 in a ratio of 1:500 revealed that aftertransfection most of the particles were taken up in the cells (see FIG.3)

11.3. Stability Towards Electrostatic Displacement:

Since the complexes of the present invention, particularly the polymersaccording to formula (I) are unique with respect to their compositionand their surface charge, unexpected results could be observed in gelshift assays. Normally, it is determined, as to whether a polymercondenses the nucleic acid and thus prevents the nucleic acid to migratein an electrical field. If a complex partner is added, which exhibits astronger affinity for the cationic polymer than the nucleic acid, thenucleic acid is displaced from the complex and again can migrate in anelectrical field. For this purpose, PEI may be used, which is known toexhibit extremely strong complexes with nucleic acid.

In a gel shift assay to examine the strength of complex binding (seeFIG. 4) it could be shown that the addition of the anionic polymerheparin can not displace the RNA from the inventive polymeric carriercargo complex because it still migrates in the gel. This indicates thatthe complex binding is so strong that a competitive complex partnercannot displace the RNA from the inventive polymeric carrier cargocomplex.

In a further a gel shift assay to examine the strength of complexbinding (see FIG. 5) it could be shown that the addition of the anionicpolymer heparin or the reducing agent DTT alone cannot display the RNAfrom the inventive polymeric carrier cargo complex with the polymeraccording to the invention (PB19). Only together they are able todisplay the RNA from the complex (lane 7). This indicates that thecomplex binding is so strong that neither a competitive complex partnernor a reducing agent can displace the RNA from the inventive polymericcarrier cargo complex.

Contrary to PEI complexes, the RNA is not released from the inventivepolymeric carrier cargo complex upon addition of heparin. Only acombination of heparin and DTT releases the RNA. It is to be noted thatDTT reduces disulfide bonds and thus destroys the conjugate. Thisimitates in vivo conditions, where the reducing conditions in the cellreleases the RNA from the complex.

In comparison thereto, gel shift assays with PEI complexes to examinethe strength of complex binding show that the addition of the anionicpolymer heparin alone cannot displace the RNA from the complex withpolymers used according to the present invention (PB22). In contrastmRNA could be readily displayed by heparin from PEI complexes (see FIG.6).

11.4. Expression of Luciferase in HeLa Cells:

The expression of luciferase in HeLa cells was determined using acomplex of a polymer according to formula (I) herein and an mRNAaccording to SEQ ID NO: 369 (mRNA coding for luciferase (luc-RNActive)with the PB19 carrier (molar ratio of RNA:PB19 1:1000, 1:500, 1:100).These formulations of mRNA coding for luciferase (luc-RNActive) with thePB19 carrier (molar ratio of RNA:PB19 1:1000, 1:500, 1:100) lead toexpression of luciferase independently of the presence of serumcontaining medium. These results are unexpected because serum containingmedium leads in general to a loss of transfection efficiency.

11.5. Expression of Luciferase In Vivo:

Expression of luciferase in BALB/c mice was determined after intradermalinjection. As can be seen in FIG. 9a , formulations of mRNA coding forluciferase (luc-RNActive) (SEQ ID NO: 369) with the PB19 polymer lead toexpression of luciferase in the dermis of female BALB/c mice. Othertransfection reagents known in the art (PEI and Lipofectamine 2000) didnot show any expression of the Luciferase protein.

Furthermore, expression of luciferase in BALB/c mice after intramuscularinjection was determined. As a result (see FIG. 9b ), formulations ofmRNA coding for luciferase (luc-RNActive) (SEQ ID NO: 369) with the PB19polymer lead to expression of luciferase in the m. tibialis of femaleBALB/c mice. Other transfection reagents known in the art (PEI andLipofectamine 2000) did not show any expression of the Luciferaseprotein.

Additionally, expression of luciferase in BALB/c mice after intradermalinjection of different formulations was determined. As a result (seeFIG. 10), formulations of mRNA coding for luciferase (luc-RNActive) (SEQID NO: 369) with two different polymers according to the presentinvention (polymers PB19 and PB48) lead to expression of luciferase inthe dermis of female BALB/c mice. The polymeric carrier cargo complexformed by a peptide according to RPC CH6R4H6C (without PEGylation) andRNA in a molar ratio of 2500:1 procedure showed no expression ofluciferase after intradermal injection of the complexed RNA.

11.6 Cytokine Stimulation in hPBMC

Cytokine stimulation, particularly hIL-6 cytokine secretion in hPBMCswas measured. As a result (see FIG. 13) it could be shown that thecomplexes according to the invention consisting of RNA (SEQ ID NO: 369)and polymers according to the invention (PB19 and PB22) do not inducethe secretion of hIL-6 in hPBMCs in contrast to complexes consisting ofRNA (SEQ ID NO: 369) and cationic peptides (H₃R₉H₃) or a combination ofcationic peptides (H₃R₉H₃) and PEGylated peptides (E9 or R9) (whichconfers in general to subsequent hydrophilic coating of pre-formednucleic acid condensates).

-   -   E9-PEG5k: HO-PEG₅₀₀₀-EEEEEEEEE    -   E9-PEG3k: HO-PEG₃₀₀₀-EEEEEEEEE    -   R9-PEG3k: HO-PEG₃₀₀₀-RRRRRRRRR    -   PB19: HO-PEG₅₀₀₀-S—(S—CHHHRRRRHHHC—S)₅—S-PEG₅₀₀₀-OH    -   PB22: HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S)₅—S-PEG₅₀₀₀-OH

Furthermore, hTNFa cytokine secretion in hPBMCs was measured. Theresults show (see FIG. 14), that the complexes according to theinvention consisting of RNA (SEQ ID NO: 369) and polymers according tothe invention (PB19 and PB22) do not induce the secretion of hTNFα inhPBMCs in contrast to complexes consisting of RNA (SEQ ID NO: 369) andcationic peptides (H₃R₉H₃) or such complexes coated with PEGylatedpeptides (E9 or R9) (which confers in general to subsequent hydrophiliccoating of pre-formed nucleic acid condensates).

-   -   E9-PEG5k: HO-PEG₅₀₀₀-EEEEEEEEE    -   E9-PEG3k: HO-PEG₃₀₀₀-EEEEEEEEE    -   R9-PEG3k: HO-PEG₃₀₀₃-RRRRRRRRR    -   PB19: HO-PEG₅₀₀₀-S—(S—CHHHRRRRHHHC—S)₅—S-PEG₅₀₀₀-OH    -   PB22: HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S)₅—S-PEG₅₀₀₀-OH

Moreover, hIFNa cytokine secretion in hPBMCs was determined in acomparison of cytokine stimulating properties of complexes according tothe invention consisting of RNA (SEQ ID NO: 369) and polymers accordingto the invention (PB19) to state of the art transfection reagents likeLipofectamin 2000 or PEI. As a result both Complexation withLipofectamin 2000 and PEI lead to a high amount of secretion of hIFNa,whereas the complex according to the invention consisting of RNA (SEQ IDNO: 369) and polymers according to the invention (PB19 500) did not.

Furthermore, hTNFa cytokine secretion in hPBMCs was measured in acomparison of cytokine stimulating properties of complexes according tothe invention consisting of RNA (SEQ ID NO: 369) and polymers accordingto the invention (PB19) to state of the art transfection reagents likeLipofectamin 2000 or PEI. As a result (see FIG. 15) Complexation withLipofectamine 2000 leads to a high amount of secretion of hTNFα, whereascomplexation with PEI or polymers according to the invention (PB19 500)did not.

1. Polymeric carrier molecule according to formula (I):L-P¹—S—[S—P²—S]_(n)—S—P³-L wherein, P¹ and P³ are different or identicalto each other and represent a linear or branched hydrophilic polymerchain, each P¹ and P³ exhibiting at least one —SH-moiety, capable toform a disulfide linkage upon condensation with component P², the linearor branched hydrophilic polymer chain selected independent from eachother from polyethylene glycol (PEG),poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethylphosphorylcholines, poly(hydroxyalkyl L-asparagine),poly(2-(methacryloyloxy)ethyl phosphorylcholine), hydroxyethylstarch orpoly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chainexhibits a molecular weight of about 1 kDa to about 100 kDa, P² is acationic or polycationic peptide or protein, having a length of about 3to about 100 amino acids, or is a cationic or polycationic polymer,having a molecular weight of about 0.5 kDa to about 30 kDa, each P²exhibiting at least two —SH-moieties, capable to form a disulfidelinkage upon condensation with further components P² or component(s) P¹and/or P³; —S—S— is a (reversible) disulfide bond; L is an optionalligand, which may be present or not, and may be selected independentfrom the other from RGD, Transferrin, Folate, a signal peptide or signalsequence, a localization signal or sequence, a nuclear localizationsignal or sequence (NLS), an antibody, a cell penetrating peptide, TAT,a ligand of a receptor, cytokines, hormones, growth factors, smallmolecules, carbohydrates, mannose, galactose, synthetic ligands, smallmolecule agonists, inhibitors or a ntagonists of receptors, or RGDpeptidomimetic analogues; and n is an integer, selected from a range ofabout 1 to 50, preferably in a range of about 1, 2, 3, 4, or 5 to 10,more preferably in a range of about 1, 2, 3, or 4 to
 9. 2. The polymericcarrier molecule according to claim 1, wherein the polymeric carriermolecule additionally contains an amino acid component (AA)_(x′) whereinx is an integer selected from a range of about 1 to 1.00.
 3. Thepolymeric carrier molecule according to claim 2, wherein the amino acidcomponent (AA)_(x) comprises an aromatic amino acid component, ahydrophilic amino acid component, a lipophilic amino acid component, aweak basic amino acid component, a signal peptide, localization signalor sequence, a nuclear localization signal or sequence, a cellpenetrating peptide, a therapeutically active protein or peptide, anantigen or an antigenic epitope, a tumour antigen, a pathogenic antigen(an animal antigen, a viral antigen, a protozoal antigen, a bacterialantigen, an allergic antigen), an autoimmune antigen, or a furtherantigen, an allergen, an antibody, an immunostimulatory protein orpeptide, an antigenspecific T-cell receptor, or another protein orpeptide suitable for a specific (therapeutic) application.
 4. Thepolymeric carrier molecule according to claim 2, wherein the amino acidcomponent (AA)_(x) occurs as a mixed repetitive amino acid component[(AA)_(x)]_(z′) wherein z is an integer selected from a range of about 1to
 30. 5. The polymeric carrier molecule according to claim 2, whereinformula (I) is modified according to formula (Ia)L-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-L, wherein x, z, S, L, AA,P¹, P² and P³ are as defined before anda+b=n, wherein n is as defined before, preferably in a range of about 1,2, 3, 4, or 5 to 10; a is an integer, selected independent from integerb from a range of about 1 to 50, preferably in a range of about 1, 2, 3,4, or 5 to 10, and b is an integer, selected independent from integer afrom a range of about 1 to 50, preferably in a range of about 1, 2, 3,4, or 5 to 10, and wherein the single components [S—P²—S] and[S-(AA)_(x)-S] occur in any order in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}.
 6. The polymeric carrier moleculeaccording to claim 1, wherein component P² is a cationic or polycationicpeptide selected from protamine, nucleoline, spermine or spermidine,poly-L-lysine (PLL), basic polypeptides, polyarginine, cell penetratingpeptides (CPPs), chimeric CPPs, Transportan, or MPG peptides,HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides,oligoarginines, members of the penetratin family, Penetratin,Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, etc., antimicrobial-derived CPPs, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, KALA,PpTG20, Proline-rich peptides, Loligomere, Arginine-rich peptides,Calcitonin-peptides, FGF, Lactoferrin, histones, VP22 derived or analogpeptides, HSV, VP22 (Herpes simplex), MAP, KALA or protein transductiondomains (PTDs, PpT620, prolin-rich peptides, lysine-rich peptides,Pep-1, L-oligomers, Calcitonin peptide(s).
 7. The polymeric carriermolecule according to claim 1, wherein the —SH-moiety of component(s) P²is provided by a cysteine.
 8. The polymeric carrier molecule accordingto claim 1, wherein component P² is selected from a peptide comprising acationic peptide of formula (IIb):Cys{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys, whereinl+m+n+o+x=8-15, and l, m, n or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 10% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native or non-native amino acidsexcept of Arg, Lys, His or Orn; and x may be any number selected from 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide.
 9. Polymeric carrier cargo complex formed of a polymericcarrier molecule according to claim
 1. 10. The polymeric carrier cargocomplex according to claim 9, wherein the nucleic acid is provided in amolar ratio of about 5 to 10000 of inventive polymeric carriermolecule:nucleic acid.
 11. The polymeric carrier cargo complex accordingto claim 9, wherein the nucleic acid is a DNA or a RNA, a coding nucleicacid, a coding DNA, a coding RNA, a coding mRNA, an siRNA, animmunostimulatory nucleic acid, an immunostimulatory CpG nucleic acid,or an immunostimulatory RNA (isRNA),
 12. The polymeric carrier cargocomplex according to claim 9, wherein the nucleic acid encodes atherapeutically active protein or peptide, an antigen, including tumorantigens, pathogenic antigens, animal antigens, viral antigens,protozoal antigens, bacterial antigens, allergic antigens, autoimmuneantigens, allergens, antibodies, immunostimulatory proteins or peptides,or antigen-specific T-cell receptors.
 13. Method of preparing apolymeric carrier molecule as defined in claim 1 comprising followingsteps a) providing at least one cationic or polycationic protein orpeptide as component P² as defined herein and/or at least one cationicor polycationic polymer as component P², and optionally at least onefurther component (AA)_(x), mixing these components, preferably in abasic milieu, preferably in the presence of oxygen or a further starterwhich leads to mild oxidation conditions, and thereby condensing andthus polymerizing these components with each other via disulfide bondsin a polymerization condensation or polycondensation to obtain arepetitive component H—[S—P²—S]_(n)—H orH{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}H; b) providing a hydrophilic polymerP¹ and/or P³, optionally modified with a ligand L and/or an amino acidcomponent (AA)_(x); c) mixing the hydrophilic polymer P¹ and/or P³according to step b) with the repetitive component H—[S—P²—S]_(n)—H orH{[S—P²—S]_(a)[S-(AA)_(x)-S]b}H obtained according to step a) in a ratioof about 2:1, and thereby typically terminating the polymerizationcondensation or polycondensation reaction and obtaining the inventivepolymeric carrier molecule according to formula (I) or (Ia); d)optionally purifying the polymeric carrier molecule obtained accordingto step c); e) optionally adding a nucleic acid as defined herein to thepolymeric carrier obtained according to step c) or d) and complexing thenucleic acid with the polymeric carrier obtained according to step c) ord) to obtain a polymeric carrier cargo complex.
 14. Pharmaceuticalcomposition or vaccine, comprising the polymeric carrier cargo complexaccording to claim 9 and optionally a pharmaceutically acceptablecarrier and/or vehicle. 15-16. (canceled)
 17. A method of treating asubject comprising administering an effective amount of a polymericcarrier cargo complex in accordance with claim 9.